Báo cáo khoa học: P-Glycoprotein is localized in intermediate-density membrane microdomains distinct from classical lipid rafts and caveolar domains potx

P-Glycoprotein is localized in intermediate-density
membrane microdomains distinct from classical lipid rafts
and caveolar domains
Galina Radeva, Jocelyne Perabo and Frances J. Sharom
Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada
In recent years, intense interest has been focussed on
the properties and biological functions of specialized
membrane domains known as lipid rafts [1,2]. Rafts
consist of cholesterol–sphingolipid-rich regions within
the plasma membrane, stabilized by interactions
between cholesterol and the long saturated acyl chains
of sphingolipids. They are thought to exist in the
liquid-ordered phase, which has properties intermedi-
ate between those of the liquid-crystalline and gel
phases [3,4]. Acylated and lipid-modified proteins are
Key words
ABC transporter; caveolin-1; detergent-
resistant membranes; lipid rafts;
P-glycoprotein
Correspondence
F. J. Sharom, Department of Molecular and
Cellular Biology, University of Guelph,
Guelph, Ontario, Canada, N1G 2W1
Fax: +519 837 1802
Tel: +519 824 4120; ext. 52247
E-mail:
(Received 11 May 2005, revised 27 July
2005, accepted 4 August 2005)
doi:10.1111/j.1742-4658.2005.04905.x
P-glycoprotein (Pgp), a member of the ATP-binding cassette (ABC) super-
family responsible for the ATP-driven extrusion of diverse hydrophobic

molecules from cells, is a cause of multidrug resistance in human tumours.
Pgp can also operate as a phospholipid and glycosphingolipid flippase, and
has been functionally linked to cholesterol, suggesting that it might be
associated with sphingolipid–cholesterol microdomains in cell membranes.
We have used nonionic detergent extraction and density gradient centrifu-
gation of extracts from the multidrug-resistant Chinese hamster ovary cell
line, CH
R
B30, to address this question. Our data indicate that Pgp is
localized in intermediate-density membrane microdomains different from
classical lipid rafts enriched in Src-family kinases. We demonstrate that
Brij-96 can selectively isolate the Pgp domains, separating them from the
caveolar and classical lipid rafts. Pgp was found entirely in the Brij-96-
insoluble domains, and only partially in the Triton X-100-insoluble
membrane microdomains. We studied the sensitivity of these domains to
cholesterol removal, as well as their relationship to GM
1
ganglioside- and
caveolin-1-enriched caveolar domains. We found that the buoyant density
of the Brij-96-based Pgp-containing microdomains was sensitive to choles-
terol removal by methyl-b-cyclodextrin. The Brij-96 domains retained their
structural integrity after cholesterol depletion while, in contrast, the Triton
X-100-based caveolin-1 ⁄ GM
1
microdomains did not. Using confocal fluor-
escence microscopy, we determined that caveolin-1 and GM
1
colocalized,
while Pgp and caveolin-1, or Pgp and GM
1

, did not. Our results suggest
that Pgp does not interact directly with caveolin-1, and is localized in inter-
mediate-density domains, distinct from classical lipid rafts and caveolae,
which can be isolated using Brij-96.
Abbreviations
ABC, ATP-binding cassette; BSS, buffered saline solution; CTB, cholera toxin B subunit; CTB–HRP, cholera toxin B–horseradish peroxidase
conjugate; DRM, detergent-resistant membranes; ECL, enhanced chemiluminescence; MbCD, methyl-b-cyclodextrin; MDR, multidrug
resistance ⁄ resistant; MEM, minimal essential medium; MRP, multidrug-resistance-associated protein; NaCl ⁄ P
i
, phosphate-buffered saline;
Pgp, P-glycoprotein.
4924 FEBS Journal 272 (2005) 4924–4937 ª 2005 FEBS
often sequestered into lipid rafts, probably as a result
of their acyl chain properties; GPI-anchored proteins
are found in the outer leaflet, and Src-family tyrosine
kinases are found in the inner leaflet. Substantial evi-
dence supports the existence of lipid raft microdomains
in model membrane systems in vitro, and in intact cells
in vivo [5–7], although there is still controversy regard-
ing their size and dynamic properties [8].
Pgp (P-glycoprotein, MDR1, ABCB1) is an energy-
dependent drug efflux pump that is a member of the
ATP-binding cassette (ABC) family of proteins [9].
Pgp decreases the intracellular concentration of a wide
variety of drugs and hydrophobic molecules by actively
transporting them across the plasma membrane, pow-
ered by ATP hydrolysis at two cytoplasmic nucleotide-
binding domains. Pgp has been proposed to act as a
drug flippase or a hydrophobic ‘vacuum cleaner’ [10].
Under normal physiological conditions Pgp is involved

in cellular detoxification leading to cell survival;
however, in cancer cells its overexpression confers a
multidrug-resistant (MDR) phenotype thus causing
chemotherapy failure [11]. An accompanying change in
many cells expressing MDR transporters, including
Pgp, is elevated levels of certain sphingolipids [12–14].
The ATPase activity of Pgp is modulated by lipids
[15–17], and its interaction with drug substrates
depends on the lipid surroundings [18]. Pgp has also
been associated with active cholesterol redistribution
across the plasma membrane [19], and cholesterol
affected its drug binding [18,20], transport and ATPase
activity [21–24]. Pgp is functional when reconstituted
into a sphingomyelin-cholesterol mixture that mimics
lipid rafts [25]; however, it can carry out both ATP
hydrolysis and drug transport in bilayers of only phos-
phatidylcholine [17,26], so cholesterol is not required
for its function. As sphingolipids and cholesterol are
both components of lipid rafts, the fine interplay
between lipid environment and Pgp function may be
linked to the membrane microdomain organization of
the protein.
Raft domains have been isolated from intact cells
based on their insolubility in cold nonionic detergents,
especially Triton X-100, and their low buoyant density
in sucrose gradients. The resulting detergent-resistant
membranes (DRM) are believed to arise from the
coalescence of smaller raft structures on the cell
surface. Caveolin-1, a 21 kDa transmembrane choles-
terol-binding protein, is the primary constituent of

invaginated plasma membrane structures called caveo-
lae. Caveolar and noncaveolar DRM microdomains
represent distinct plasma membrane regions [27,28].
Caveolin-1, GM
1
ganglioside and cholesterol are
believed to be hallmarks of caveolae which are distinct
from the classical lipid rafts that are enriched in GPI-
anchored proteins, cholesterol and GM
1
, but do not
contain caveolins [29]. Up-regulation of caveolin-1 and
caveolae has been observed in MDR cells expressing
Pgp, suggesting a functional link between them [30,31].
Interestingly, Pgp was reported to appear in the low
density membrane fractions in Triton X-100 extracts
[32], as well as in detergent-free cell extracts [21].
Demeule and coworkers found that Pgp was contained
in the caveolae in MDR cells and blood–brain barrier
endothelial cells [33,34]. In contrast, Hinrichs et al.
determined that Pgp was localized in the noncaveolar
rafts [35], while flow cytometry and confocal microsco-
py showed that a substantial fraction of Pgp was asso-
ciated with lipid rafts and the cytoskeleton in human
colon carcinoma cells [36].
We recently reported that the nonionic detergents
Brij-96 and Triton X-100 isolated different lipid raft
microdomains from rat basophilic leukemia (RBL-
2H3) cells [37]. We therefore employed these detergents
to investigate the microdomain localization of Pgp in

the MDR cell line CH
R
B30. In the present work, we
showed that this ABC transporter is localized in inter-
mediate-density membrane microdomains that are dis-
tinct from caveolar domains and Src kinase-containing
classical lipid rafts. We also showed that these
domains are differentially extracted by Brij-96, but not
by Triton X-100. In addition, we found that Brij-96
segregates caveolar domains from Src kinase-based
classical lipid rafts, leading to distinct sets of fractions
containing each class of raft. Triton X-100 extraction
apparently leads to the copartitioning of different types
of membrane microdomains ino a common pool. Tri-
ton X-100 rafts are disrupted by cholesterol removal,
whereas the Brij-96 rafts change their buoyant density,
but maintain their structural integrity.
Results
Pgp is localized in intermediate-density
membrane microdomains
DRM are commonly isolated by cold nonionic deter-
gent extraction followed by sucrose density centrifuga-
tion. We previously showed that Brij-96 and Triton
X-100 isolate lipid rafts with different physical and
biochemical properties from RBL-2H3 cells [37]. In
this work, we used a similar approach to investigate
the membrane domain localization of Pgp. Brij-96 or
Triton X-100 extracts of CH
R
B30 cells expressing Pgp

were subjected to sucrose density gradient flotation,
and the distribution of Pgp among the fractions was
determined by western blotting.
G. Radeva et al. P-glycoprotein in intermediate-density microdomains
FEBS Journal 272 (2005) 4924–4937 ª 2005 FEBS 4925
Triton X-100 extraction of CH
R
B30 cells yielded a
bimodal distribution for Pgp (Fig. 1A, right panel, top
row). This type of distribution is typical for the protein
constituents of classical lipid rafts, such as GPI-
anchored proteins and the Src tyrosine kinases. A
significant amount of Pgp was observed in the low-
density lipid raft fractions 4–6, while the majority of
the transporter remained in the high-density fractions
9–11. However, in the density gradient fractions from
Brij-96 extracts, Pgp displayed a continuous distribu-
tion in fractions 2–10, peaking in fractions 5 and 6
(Fig. 1A, left panel, top row). This pattern of Pgp par-
titioning along the sucrose density gradient is quite
unlike the picture observed for the known constituents
of lipid rafts, such as GPI-anchored proteins or Src
kinases. In our previous work, we showed that under
the same conditions, the lipid raft protein Thy-1 of
RBL-2H3 cells is concentrated entirely in the lowest
density lipid raft fractions 2–4 [37]. We have therefore
termed the fractions in which Pgp is incorporated, as
intermediate-density fractions. The data indicated that
all of the Pgp in CH
R

B30 cells is localized in Brij-96-
based domains that are completely resistant to solubili-
zation with this detergent. Furthermore, Pgp is only
partially located in the Triton X-100-resistant DRM,
and about half of it can be solubilized by extraction
with this detergent.
Pgp is an N-glycosylated protein [38], and because
glycosylation may affect the membrane domain local-
ization of proteins, we examined whether the glycosy-
lation status of Pgp had any bearing on its distribution
in the density gradient after extraction using Brij-96.
To address this, we used the CH
R
PHA
R
cell line (a
lectin-resistant variant of the parental line used to
derive CH
R
B30), which is deficient in N-linked glyco-
sylation. The results presented in Fig. 1A show that
the profile for Pgp localization in the sucrose density
gradient is very similar when either cell line is used
with each of the detergents (compare first and second
rows). We conclude that glycosylation does not play
a role in the partitioning of Pgp into intermediate-
density microdomains.
A
B
Fig. 1. Sucrose density gradient partitioning

of P-glycoprotein (Pgp) and markers of clas-
sical lipid rafts. (A) CH
R
B30 cells or
CH
R
PHA
R
cells (second row only) were
lysed in either 0.5% (w ⁄ v) Brij-96 or 1%
(w ⁄ v) Triton X-100 at 4 °C, and postnuclear
lysates were fractionated by ultracentrifuga-
tion on a discontinuous sucrose gradient. A
total of 13 fractions was collected from the
top of the gradient tube and an aliquot from
each fraction was run on SDS ⁄ PAGE. Separ-
ated proteins were transferred to a nitrocel-
lulose membrane and the presence of Pgp,
Yes, caveolin-1 (Cav-1), and CD71 was
observed by western immunoblot analysis
and enhanced chemiluminescence (ECL)
detection, as described in the Experimental
procedures. (B) CH
R
B30, RBL-2H3 and Jur-
kat cells were lysed in Triton X-100 at 4 °C.
Lysates were precleared by centrifugation at
10 000 g for 5 min. An aliquot from each
extract was run on SDS ⁄ PAGE, and the
separated proteins were transferred to a

nitrocellulose membrane and analyzed for
Src-family kinases (Lck, Lyn, and Yes) by
western immunoblot analysis and ECL.
P-glycoprotein in intermediate-density microdomains G. Radeva et al.
4926 FEBS Journal 272 (2005) 4924–4937 ª 2005 FEBS
Comparison of the density gradient partitioning
of Pgp and markers of classical lipid rafts
CH
R
B30 cells have little or no expression of the most
common GPI-anchored proteins, such as Thy-1, alka-
line phosphatase, decay accelerating factor, etc. We
therefore employed Src family tyrosine kinases for the
identification of classical lipid rafts and comparison
with the intermediate-density Pgp membrane domains.
First, we determined which members of the Src tyro-
sine kinase family are expressed in CH
R
B30 cells, using
extracts from RBL-2H3 and Jurkat cells as positive
controls (Fig. 1B). Of the three proteins we tested for
(Lck, Lyn and Yes), Lyn was expressed only in RBL-
2H3 cells, and Lck only in Jurkat cells, whereas Yes
was seen in both of these cell lines. In CH
R
B30 cells
we found that only Yes kinase was detectable.
Next, we investigated how the distribution of Yes kin-
ase along the density gradient compared with that of
Pgp. When Triton X-100 was used, Yes kinase resided

in almost the same fractions as Pgp. A portion of Yes
kinase partitioned into the low-density sucrose fractions
3–6, while a significant amount (about half) remained
in the high-density fractions 10–13 (Fig. 1A, third row,
right panel). When Brij-96 was used, Pgp and Yes kinase
did not copartition, as determined by density centrifuga-
tion (Fig. 1A, third row, left panel). Yes kinase was
localized exclusively in the lowest-density fractions 1–4.
This localization is similar to that obtained for another
Src-family tyrosine kinase, Lyn, whose sucrose density
gradient partitioning was examined in RBL-2H3 cells
following extraction with Brij-96 [37]. The data presen-
ted in Fig. 1A indicate that Pgp is localized in mem-
brane microdomains that are distinct from classical lipid
rafts containing Src-family tyrosine kinases. The mem-
brane microdomains containing Pgp displayed an inter-
mediate density in the sucrose gradient when Brij-96 was
used. They can be separated from classical lipid rafts if
extracted with Brij-96, but not with Triton X-100.
The total protein content of each fraction was meas-
ured by the bicinchoninic acid assay, as shown in
Fig. 2 (lower panel). Both detergents solubilized the
majority of cellular proteins, leaving them in the high-
density soluble fractions 11–13. Brij-96 extraction
resulted in small, but detectable, amounts of protein in
the low density fractions, whereas Triton X-100 extrac-
tion resulted in virtually no protein in these fractions.
Relationship of the intermediate-density
Pgp-containing microdomains to caveolae
It is well-documented that at least two types of deter-

gent-insoluble membrane microdomains exist. The
first class encompasses the classical lipid rafts (or
noncaveolar lipid rafts), which contain GPI-anchored
proteins and Src-family kinases, while the other class
represents the caveolar raft microdomains, with cave-
olin as a hallmark protein. We examined the possible
relationship between the intermediate-density mem-
brane structures in which Pgp is found, and caveolae
structures, by assessing copartitioning of Pgp and
caveolin-1 in the sucrose density gradient fractions.
Caveolin-1 was concentrated in fractions 4–8 in both
of the detergent extracts, although in the case of Tri-
ton X-100 there was tailing out to fractions 11–12
(Fig. 1A, fourth row). Importantly, the localization of
caveolin-1 displayed a significant overlap with that of
Pgp in both the low-density fractions from Triton
X-100 extracts and in the intermediate-density frac-
tions from Brij-96 extracts (Fig. 1A, compare the first
row with the third row).
These results suggest two interesting possibilities.
First, Brij-96 appears to differentially isolate the
caveolar (fractions 4–8, caveolin-1 marker) from
Fig. 2. Protein and GM
1
profile of Triton X-100 and Brij-96 sucrose
density gradients. Post-nuclear lysates of detergent extracts of
CH
R
B30 cells were run on sucrose gradients, and the gradient frac-
tions were assayed for the distribution of total protein and GM

1
ganglioside, as described in the Experimental procedures. The pro-
tein content is shown for a 20 lL aliquot of each gradient fraction
from 5–10 · 10
8
cells lysed in 1 mL of buffer, and the activity of
cholera toxin B–horseradish peroxidase conjugate (CTB–HRP) in a
50 lL aliquot of each gradient fraction from 2–3 · 10
8
cells is indi-
cated. Data are displayed as the mean ± range; where error bars
are not visible, they are contained within the symbols.
G. Radeva et al. P-glycoprotein in intermediate-density microdomains
FEBS Journal 272 (2005) 4924–4937 ª 2005 FEBS 4927
noncaveolar (fractions 1–4, Yes marker) lipid rafts.
Such a distribution was not observed in the sucrose
density fractions from the Triton X-100 extracts. The
observed differences were not an artefact of the deter-
gent used because the integral membrane protein,
CD71 (transferrin receptor), was fully solubilized by
both Brij-96 and Triton X-100 (Fig. 1A, bottom row).
Second, the Pgp distribution profile partially over-
lapped with that of caveolin-1, but not with that of
Yes kinase, when Brij-96 was used. This observation
suggests that the intermediate-density fractions con-
taining Pgp might represent caveolar membrane struc-
tures. Such an idea is in agreement with previous
reports suggesting a close interaction between Pgp and
caveolin-1 [33,34]. However, this observation does not
necessarily signify molecular colocalization of Pgp and

caveolin-1. For example, all three proteins – Pgp, Yes,
and caveolin-1 – segregate into membrane domains of
similar density when Triton X-100 is used to prepare
the cell extracts, but exhibit different distribution
patterns in the case of Brij-96 detergent extraction
(Fig. 1A). The potential colocalization of Pgp and
caveolin-1 was therefore further examined directly by
confocal fluorescence microscopy and immunoprecipi-
tation experiments, as described below.
Identification of lipid raft microdomains by
detection of lipid raft-associated GM
1
ganglioside
GM
1
ganglioside is a known marker of both classical
lipid rafts and caveolae. This glycosphingolipid has
been shown to cofractionate not only with markers of
various detergent-insoluble microdomains (such as
caveolae, GPI-anchored protein-enriched rafts, and
glycosphingolipid-enriched domains), but also to colo-
calize with caveolin-1 [39]. We employed a cholera
toxin B–horseradish peroxidase conjugate (CTB–HRP)
enzyme assay to identify the fractions into which lipid
raft-associated GM
1
partitions (Fig. 2, upper panel).
For Triton X-100, these were fractions 5, 6, and 7. In
the density gradient of Brij-96 extracts, GM
1

was
detected in fractions 2–5. This pattern is very similar
to that observed for GM
1
in RBL-2H3 cells [37]; how-
ever, the peak seen for GM
1
localization in the
CH
R
B30 gradient fractions is somewhat broader. The
gradient partitioning of GM
1
(Fig. 2, upper panel) par-
tially overlaps with the Yes kinase classical lipid rafts
fractions on the one hand, and with the caveolin-1-
enriched raft fractions on the other (Fig. 1A). This
broader profile can be explained by the fact that GM
1
is a constituent of both classical lipid rafts and caveo-
lae. The high level of GM
1
apparently present in the
high density fractions of the gradient in Fig. 2 (upper
panels), which is not observed in the dot-blots (Fig. 6,
panel B) is probably spurious. This was also reported
by Blank et al. [40], and could arise from the presence
of soluble HRP-like activity in the cells.
Examination of Pgp and caveolin-1 localization
by confocal fluorescence microscopy and

immunoprecipitation
We wanted to determine whether the intermediate-
density fractions containing Pgp in the Brij-96 extract
represent caveolar membrane microdomains. Demeule
et al. reported that Pgp and caveolin-1 coimmunopre-
cipitated in extracts from Pgp-expressing CH
R
C5 cells
and brain capillary membranes [33]. We tested the
coimmunoprecipitation of Pgp and caveolin-1 using
the pooled lipid raft fractions from the Brij-96 and
Triton-100 extracts. However, we were unable to
detect coimmunoprecipitation between the two pro-
teins under these conditions. We decided therefore to
examine their potential association using total cell
extracts because these would contain the entire pool
of Pgp and caveolin-1. Cell extracts were prepared
using various lysis buffer conditions. Combinations of
different detergents were used to establish whether the
choice of detergent plays a role in the observation of
coimmunoprecipitation of the two proteins. Hinrichs
and coworkers had already reported a weak associ-
ation of multidrug resistance-associated protein 1
(MRP1) with caveolin-1 when Lubrol was used, but
saw no such association in the presence of Triton X-
100 [35]. In our experiments, all buffers contained
sufficient detergent to disrupt the vesicles previously
observed to exist in the DRM fractions [37]. Other-
wise, a false impression of coimmunoprecipitation
would be obtained if the two proteins were simply

located in the same vesicular structure. Under these
conditions, only a very faint band of Pgp was seen in
the caveolin-1 immunoprecipitates (Fig. 3A, top), but
no signal for caveolin-1 was detected in the Pgp
immune complexes (Fig. 3A, bottom), suggesting that
there is no significant coimmunoprecipitation between
the two proteins. A signal for caveolin-1 in Pgp immu-
noprecipitates was seen only after prolonged overnight
exposure (Fig. 3B, bottom), while an enhanced Pgp
band was seen in the caveolin-1 immunoprecipitates
when the film was overexposed (Fig. 3B, top). We sug-
gest that either a very small fraction of the two pro-
teins is associated with each other, or that they are
located close together in the membrane, but not
directly interacting with one another. This result
agrees with the results of confocal immunofluorescence
analysis (see below) and is in accordance with the
P-glycoprotein in intermediate-density microdomains G. Radeva et al.
4928 FEBS Journal 272 (2005) 4924–4937 ª 2005 FEBS
work of Hinrichs et al. [35], who also reported no
coimmunoprecipitation between the two proteins in
2780AD human ovarian carcinoma cells.
We further examined the possible cellular colocaliza-
tion of the two proteins by confocal fluorescence
microscopy. We first examined the colocalization of
GM
1
and caveolin-1 in CH
R
B30 cells. The individual

staining patterns for GM
1
and caveolin-1 were very
similar; bright punctuate spots were observed, mainly
on the plasma membrane (Fig. 4A,B). When the sig-
nals from the two dyes, Alexa
488
and Alexa
594
, were
superimposed, there were several areas of clear over-
lap, as indicated by the yellow colour (Fig. 4C, over-
lay). This observation indicates a close colocalization
of GM
1
and caveolin-1 in some regions of the cell,
probably in the caveolar raft domains. Next we investi-
gated the localization of Pgp and GM
1
(Fig. 4D,E).
We found that the cellular localization patterns of Pgp
and GM
1
were distinct and did not overlap, indicating
that the two molecules do not directly interact
(Fig. 4F). When the localization of caveolin-1 and Pgp
was compared, the signals for these proteins were,
once again, very distinct (Fig. 4G,H). Both caveolin-1
and Pgp maintained the pattern described above. Pgp
displayed staining at the plasma membrane but also

A
B
Fig. 3. Immunoprecipitation of P-glycoprotein (Pgp) and caveolin-1.
(A) Lanes 1 and 6, Brij-96 extracts; lanes 2 and 7, Triton X-100
extracts; lanes 3 and 8, Nonidet P-40 ⁄ Triton X-100 ⁄ octylglucoside
extracts; lanes 4 and 9, Brij-96 ⁄ radioimmunoprecipitation assay
(RIPA) extracts; lanes 5 and 10, Triton X-100 ⁄ RIPA extracts. One
microgram of each anti-Pgp or anti-(caveolin-1) immunoglobulin was
added to 500 lL of cell extracts. Immune complexes were collec-
ted on Protein-A–Sepharose beads and washed twice in the appro-
priate buffer. The immunoprecipitated proteins were extracted in
Laemmli’s sample buffer. One half of each immunoprecipitation
sample was run on 7.5% nonreducing SDS ⁄ PAGE for Pgp analysis
(A and B top). The other half of each sample was run on 12% non-
reducing SDS ⁄ PAGE for caveolin-1 analysis (A and B, bottom). Sep-
arated proteins were transferred to a nitrocellulose membrane and
analysed by western immunoblot (IB) analysis and enhanced chemi-
luminescence. The film exposure time in (A) was 5 min; (B) is an
overnight exposure of (A).
GM
1
Cav-1
Cav-1
overlay
GM
1
Pgp overlay
Pgp
overlay
A

BC
D
EF
G
H
I
Fig. 4. Confocal fluorescence microscopy analysis of P-glycoprotein
(Pgp) and caveolin-1 localization. CH
R
B30 cells grown in monolayer
culture were first labelled with cholera toxin B–horseradish peroxi-
dase conjugate (CTB–HRP), as described in the Experimental
procedures. Cells were then fixed in 4% paraformaldehyde in phos-
phate-buffered saline (NaCl ⁄ P
i
), pH 7.4, permeabilized in 0.1% (v ⁄ v)
Triton X-100 and blocked in 5% (w ⁄ v) skim milk. Pgp and caveolin-1
(Cav-1) proteins were detected with mouse and rabbit immunoglob-
ulin, respectively, and localization was revealed with anti-species
immunoglobulin conjugated to either Alexa
488
(green) or Alexa
594
(red) fluorophores. CTB–HRP was conjugated to the Alexa
488
fluoro-
phore. The overlay image was produced by superimposing the
image from the green and red channels, using LCS Lite software.
G. Radeva et al. P-glycoprotein in intermediate-density microdomains
FEBS Journal 272 (2005) 4924–4937 ª 2005 FEBS 4929

showed intracellular and perinuclear staining. When an
overlay image from the two proteins was produced, no
association between Pgp and caveolin-1 was observed,
as indicated by the absence of any yellow colour
(Fig. 4I).
Cholesterol distribution in sucrose density
fractions following treatment of CH
R
B30 cells
with methyl-b-cyclodextrin
Cholesterol is a component of classical lipid rafts that
is proposed to be required for their structural integrity.
The removal of cholesterol by various agents often
leads to the disruption of microdomain structures,
which is manifested by detergent extraction of mole-
cules residing there or by altered activity of signalling
components. We have demonstrated previously that
Pgp is localized in intermediate-density membrane
microdomains, which are distinct from classical lipid
rafts and caveolar rafts. Our next step was to examine
whether cholesterol plays a role in the formation and
stabilization of these domains. One commonly used
agent for the depletion of cholesterol is methyl-b-cyclo-
dextrin (MbCD). We tested various concentrations of
MbCD (5–50 mm) with CH
R
B30 cells, and found that
treatment with 20 mm MbCD for up to 1 h produced
the maximal cholesterol depletion while still preserving
cell viability (G. Radeva & S. J. Sharom, unpublished

data).
We measured the cholesterol content of the density
gradient fractions of extracts from untreated CH
R
B30
cells and from cells treated with MbCD, and found
that cholesterol was effectively removed from the lipid
raft fractions obtained using both detergents (Fig. 5).
In untreated cells, cholesterol displayed a bimodal dis-
tribution profile when the extracts were prepared with
Triton X-100. One peak of cholesterol was seen
around fractions 4–6 and another in fractions 9–13
(Fig. 5, bottom). This pattern corresponds to the lipid
raft marker protein distribution for this detergent
(Fig. 1A). This cholesterol distribution pattern is also
similar to that reported in our previous study in the
RBL-2H3 cell line [37]. When CH
R
B30 cells were trea-
ted with MbCD and then extracted with Triton X-100,
the cholesterol content was dramatically reduced in
lipid raft fractions 4–6, and to a lesser extent in
fractions 9–13. When lipid rafts were isolated using
Brij-96, cholesterol partitioned into a single peak
exclusively in fractions 1–5, which falls into the region
where protein markers of classical lipid rafts segregate
(Fig. 1A). Upon treatment with MbCD, cholesterol
was significantly depleted from these fractions (Fig. 5,
top).
Effect of cholesterol depletion on Pgp, caveolin-1

and GM
1
distribution in the sucrose density
gradient
After we determined that cholesterol was effectively
depleted from lipid raft fractions upon treatment with
MbCD, we examined whether cholesterol removal had
an effect on the distribution of Pgp, caveolin-1 and
GM
1
in the sucrose density gradient. We found that
Pgp located in the low-density raft fractions 4–6 in
untreated cells was shifted slightly towards the high-
density fractions when cells were extracted with Triton
X-100 (Fig. 6A, right panel). In addition, more Pgp
appeared in the high-density soluble fractions relative
to those of low density upon cholesterol depletion.
Fig. 5. Cholesterol distribution in sucrose density fractions follow-
ing treatment of CH
R
B30 cells with methyl-b-cyclodextrin (MbCD).
CH
R
B30 cells treated with 20 mM MbCD (grey bars) or untreated
control cells (black bars) were lysed in either 0.5% (w ⁄ v) Brij-96 or
1% (w ⁄ v) Triton X-100. Post-nuclear cell extracts were then run on
sucrose gradients, and the separated gradient fractions were
assayed for the distribution of cholesterol as described in the
Experimental procedures. The cholesterol content is shown for the
entire gradient fraction from 1–2 · 10

8
cells lysed in 300 lL of buf-
fer, as the mean ± range. The results shown in Figs 5 and 6 were
obtained using the same set of gradient fractions.
P-glycoprotein in intermediate-density microdomains G. Radeva et al.
4930 FEBS Journal 272 (2005) 4924–4937 ª 2005 FEBS
Interestingly, in Brij-96 extracts, there was a significant
shift in the Pgp distribution after cholesterol depletion.
Pgp was located primarily in fractions 6–11 following
cholesterol depletion, as compared to fractions 3–10
for the untreated cells (Fig. 6A, left panel). This indi-
cates a substantial change in the buoyant density of
the intermediate-density fractions upon cholesterol
depletion. GM
1
distribution also shifted towards the
higher-density fractions of the gradient after choles-
terol was removed (Fig. 6B), for rafts isolated
using Brij-96. However, no Pgp was found in the high-
density fractions, suggesting that these microdomains
retain their structural integrity on cholesterol deple-
tion. However, when rafts were extracted with Triton
X-100 following cholesterol removal, GM
1
was not
only shifted to slightly higher density in the raft frac-
tions but could also be seen in the high-density soluble
fractions 10–11 (Fig. 6B). Caveolin-1 also showed dif-
ferent behaviour in the two detergent extracts. The
protein shifted towards the higher-density fractions

upon treatment with MbCD in Brij-96 cell extracts
(Fig. 6C, left panel). However, if cholesterol-depleted
cells were extracted with Triton X-100, a large fraction
of the caveolin-1 partitioned into the high-density sol-
uble fractions 10–12 (Fig. 6C, right panel), while the
remaining protein remained localized in fractions 5–7.
This behaviour is similar to that seen for GM
1
under
the same conditions. These results suggest that the
domains in which GM
1
and caveolin-1 are located
prior to cholesterol depletion, corresponding to the
low density fractions, require cholesterol for their sta-
bilization and are disrupted when it is removed.
Discussion
The lipid raft hypothesis proposes the existence of
discrete microdomains in cellular plasma membranes,
which arise from the specific interactions of sphingo-
lipids, glycosphingolipids and cholesterol. Pgp has
recently been proposed to mediate active cholesterol
redistribution in the plasma membrane [19]. It has also
been reported that MDR cells display differential
expression and accumulation of glycosphingolipids
[12–14]. These observations were suggestive of a speci-
fic membrane domain organization for Pgp, prompting
us to examine this issue using techniques commonly
A
B

C
Fig. 6. Effect of cholesterol removal on the
distribution of P-glycoprotein (Pgp), GM
1
and caveolin-1 in the sucrose density gradi-
ent. CH
R
B30 cells treated with 20 mM
methyl-b-cyclodextrin (MbCD) or untreated
control cells were lysed in either 0.5%
(w ⁄ v) Brij-96 or 1% (w ⁄ v) Triton X-100.
Post-nuclear lysates were fractionated on a
5–30% discontinuous sucrose gradient, and
13 fractions were collected. An aliquot from
each fraction was run on SDS ⁄ PAGE, and
the separated proteins were transferred to a
nitrocellulose membrane and analysed for
Pgp (A) and caveolin-1 (C) by western
immunoblot (IB) analysis and enhanced
chemiluminescence (ECL) detection. GM
1
(B) detection was carried out by dot-blot
analysis. The results in Figs 5 and 6 were
obtained using the same set of gradient
fractions. Note that these experiments were
carried out under somewhat different condi-
tions from Fig. 1; as a result, the distribution
of caveolin-1 in the sucrose gradient is
slightly narrower.
G. Radeva et al. P-glycoprotein in intermediate-density microdomains

FEBS Journal 272 (2005) 4924–4937 ª 2005 FEBS 4931
employed to study lipid rafts, namely cold nonionic
detergent extraction and sucrose density centrifugation.
We have recently demonstrated that in RBL-2H3 cells,
Brij-96 and Triton X-100 isolate DRM with different
physical and biochemical properties [37]. Here we
present evidence that Pgp is localized in intermediate-
density membrane microdomains that are completely
insoluble in Brij-96, but partially soluble in Triton
X-100. Other ABC transporter proteins appear to
reside in Lubrol WX-resistant domains, but not in Tri-
ton X-100-resistant domains [35,41,42]. Two yeast
ABC transporters have been reported to be involved in
trafficking cholesterol specifically from lipid raft micro-
domains in the plasma membrane to the endoplasmic
reticulum, thus facilitating exogenous sterol uptake
into the cell [43].
We used Yes kinase as a marker for classical lipid
rafts and found that Pgp does not segregate with this
protein upon extraction with Brij-96. Thus, the inter-
mediate-density domains containing Pgp generated
using Brij-96 are distinct from classical lipid rafts.
There have been other reports of the existence of non-
classical rafts. For example, hepatitis C core protein
was associated with DRM that did not colocalize with
GM
1
or caveolin-1, and Drobnik et al. found that the
GPI-anchored molecules CD14 and CD55 did not
colocalize with ABCA1 after isolation of Lubrol rafts

[41]. In polarized HepG2 cells, Lubrol WX-insoluble
and Triton X-100-insoluble domains with differing
properties were functionally linked to distinct traffick-
ing pathways in the apical targeting of proteins [42].
Lubrol WX-based rafts were also described where var-
ious ABC proteins were entirely recovered in the
Lubrol-insoluble fractions and only partially (or not at
all) in the Triton X-100-insoluble fractions [35,41,42].
These observations are consistent with our findings
that Pgp extracted from CH
R
B30 cells is partially solu-
bilized by Triton X-100 but is completely resistant to
Brij-96 solubilization. We previously showed that the
degree of enrichment of microdomain constituents in
various regions of the density gradient depends on the
ratio of cell number to detergent [37]. The observed
differences in microdomain localization of ABC pro-
teins might therefore reflect variations in the amount
of cellular starting material relative to detergent.
Indeed, we found it necessary to double the
cell : detergent ratio when using CH
R
B30 cells, com-
pared to RBL-2H3 cells, in order to detect the protein
constituents of lipid rafts in the sucrose gradient frac-
tions.
Proteins that partition into lipid rafts are generally
those with lipid modifications, such as GPI-anchored
proteins, or acylated proteins that are members of the

Src tyrosine kinase family, while many integral mem-
brane proteins appear to be excluded. Recent data,
including the present work, points out that multispan-
ning proteins of the ABC transporter superfamily may
display lipid raft domain localization [35,41,42]. Cyc-
lic-nucleotide-gated channels also appear to be targeted
to lipid rafts [44]. It is conceivable that some proteins
with transport functions may be organized into mem-
brane microdomains, probably together with regula-
tory molecules, thus providing an additional level of
control over the entry and exit of their substrates.
One of the apparent differences between the Brij-96
and Triton X-100-insoluble microdomains in CH
R
B30
cells is their buoyant density, which is determined by
lipid composition and protein content. Cholesterol
is often required for maintaining lipid rafts but may
also modulate Pgp catalytic and transport activity
[19,21,36]. We found that the Brij-96-insoluble mem-
branes contain most of the cellular cholesterol, while
the Triton X-100-insoluble domains comprise only a
fraction of total cholesterol, the remainder of which is
located in the high-density soluble fractions. However,
in RBL-2H3 cells, most of the cholesterol in Triton
X-100 extracts was detected in the low-density frac-
tions [37], indicating that cell-specific differences exist
in raft microdomain detergent solubility. Drobnik
et al. also detected a lower percentage of cellular cho-
lesterol in the low-density fractions of Triton X-100

lysates, as compared to high-density fractions, in
human skin fibroblasts but not in monocytes [41].
Their data corroborate our current findings and sug-
gest that the ratio of cholesterol in the low-density vs.
high-density fractions in Triton X-100 extracts is a cell
type-specific phenomenon.
Upon depletion of cellular cholesterol by Mb CD
treatment, the Pgp-containing intermediate-density
domains isolated using Brij-96 showed a shift to higher
buoyant densities. However, the domains retained their
structural integrity as no Pgp was solubilized into the
high-density fractions. Cholesterol may not be neces-
sary for the maintenance of some types of membrane
microdomains, for example those containing K-ras [45]
and galectin-4 [46]. In contrast, the Pgp-containing Tri-
ton X-100 microdomains remaining after cholesterol
depletion showed only a small shift in buoyant density.
However, a significant fraction of these domains
appeared to have been disrupted, so that more Pgp
appeared in the soluble high-density fractions, indica-
ting a strong cholesterol requirement for maintenance
of their integrity. This finding also suggests that the
reason only a fraction of the cellular Pgp is observed
in the Triton X-100-insoluble fractions may be that
cholesterol is removed from these domains upon
P-glycoprotein in intermediate-density microdomains G. Radeva et al.
4932 FEBS Journal 272 (2005) 4924–4937 ª 2005 FEBS
detergent treatment. Indeed, a larger proportion of cel-
lular cholesterol is seen in the high-density fractions of
Triton X-100 extracts, in contrast to Brij-96 extracts.

Interestingly, the effect of cholesterol removal on GM
1
and caveolin-1 distribution was far more profound in
the Triton X-100 rafts than in the Brij-96 rafts. The
GM
1
- and caveolin-1-containing domains not only
showed a shift to higher density, but significant
amounts of GM
1
and caveolin-1 were also detected in
fractions 10–12, indicating that cholesterol depletion
leads to their solubilization. GM
1
and caveolin-1 do
not behave identically to Pgp, probably because the
critical level of cholesterol required to maintain their
raft association is different, so their sensitivity to cho-
lesterol depletion varies.
Possible associations between Pgp, GM
1
and caveo-
lin-1 were investigated by confocal fluorescence micros-
copy. Clear colocalization was seen between caveolin-1
and GM
1
, consistent with fact that caveolar fractions
are enriched in GM
1
[39]. However, we did not observe

colocalization between Pgp and caveolin-1, or Pgp and
GM
1
. Our findings agree with those of Hinrichs et al.
who reported that the ABC transporter MRP1 does
not colocalize with caveolin-1 and is enriched in non-
caveolar detergent-insoluble domains [35]. However,
Demeule et al. reported coimmunoprecipitation of Pgp
and caveolin-1 in CH
R
C5 cells and brain endothelial
cells [33,34]. We were unable to see any interaction
between Pgp and caveolin-1 by coimmunoprecipitation
under conditions where the raft vesicles are solubilized
by detergent. It is therefore possible that Pgp and cave-
olin-1 are localized in neighbouring raft domains at the
plasma membrane and copartition into the same DRM
after detergent extraction, but there is no direct, strong
association between them. Alternatively, preservation
of their interaction is highly dependent on the ratio of
cell lipid ⁄ protein : detergent employed during extrac-
tion. The association between Pgp and caveolin-1 may
be cell type-specific, but the CH
R
B30 cell line used in
this study was derived from CH
R
C5, so this seems
unlikely.
Lipid raft microdomains are proposed to exist in the

more highly ordered l
o
phase, compared to the bulk
membrane lipids, which are in the liquid-disordered l
d
phase. Work with the fluorescent probe, merocyanine
540, showed that increasing Pgp expression in MDR
cells correlated with an increase in the packing density
of the plasma membrane outer leaflet, relative to that of
the drug-sensitive parent [47], perhaps reflecting larger
numbers of raft microdomains containing Pgp. Unlike
many membrane transporters, which often cease to
function in rigid gel phase bilayers, the rate of Pgp-
mediated drug transport remained high in the gel phase
[26], suggesting that ordered microdomains may be help-
ful to the function of the protein. Pgp-mediated ATP
hydrolysis was also efficient in the gel phase, with a
lower activation energy, E
act
, than in the liquid-crystal-
line phase [17]. The intermediate density microdomains
in which Pgp is located may therefore provide a suitable
environment for the protein to function optimally.
Experimental procedures
Materials
The anti-Pgp monoclonal antibody, C219, was supplied by
ID Laboratories (London, ON, Canada). Anti-Lyn, anti-
Yes, anti-Lck, anti-caveolin-1 and anti-CD71 (transferrin
receptor) mouse monoclonal antibodies were purchased
from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

CTB–HRP, MbCD, Protein A–agarose, Protein G–agarose,
phenylmethanesulfonyl fluoride, Nonidet P-40, DNase,
pepstatin A, n-octylglucoside and leupeptin were purchased
from Sigma Chemical Co. (St Louis, MO, USA).
HRP-labelled goat anti-rabbit and goat anti-mouse immu-
noglobulin were purchased from Jackson Immunoresearch
Laboratories (Mississauga, ON, Canada). Triton X-100 was
supplied by Roche Diagnostics (Laval, QC, Canada), Brij-
96 was obtained from Fluka (Oakville, ON, Canada), and
SDS was purchased from Fisher Scientific (Whitby, ON,
Canada).
Cells
The highly MDR Chinese hamster ovary cell line, CH
R
B30,
and a glycosylation deficient lectin-resistant variant,
CH
R
PHA
R
, were as described previously [48]. Cells were
grown as monolayers in a-minimal essential medium
(a-MEM) containing 10% (v ⁄ v) fetal bovine serum (Hy-
clone, Logan, UT, USA) supplemented with 2 mm glutamine
and 2 mm penicillin ⁄ streptomycin, at 37 °C in a humidified
atmosphere of 5% (v ⁄ v) CO
2
in the presence of 30 l g Æ mL
)1
colchicine. Typically, cells were harvested using 0.25% (w ⁄ v)

trypsin or 5 mm EDTA in phosphate-buffered saline
(NaCl ⁄ P
i
, pH 7.4). The RBL-2H3 cell line was cultured as
described previously [37]. Jurkat cells were grown using the
same culture medium and conditions as CH
R
B30 cells.
Isolation of lipid raft microdomains using sucrose
gradient centrifugation
Lipid rafts were isolated from either freshly harvested or
frozen cells, using Triton X-100 or Brij-96, as described pre-
viously for RBL-2H3 cells [37]. About 5–10 · 10
8
cells
(200–250 lL cell pellet) were washed twice in NaCl ⁄ P
i
,
pH 7.4 or Tris-buffered saline (TBS; 25 mm Tris ⁄ HCl,
140 mm NaCl, pH 7.5) and then treated on ice with 1 mL
G. Radeva et al. P-glycoprotein in intermediate-density microdomains
FEBS Journal 272 (2005) 4924–4937 ª 2005 FEBS 4933
of lysis buffer consisting of 0.5% (w ⁄ v) Brij-96 or 1%
(w ⁄ v) Triton X-100 in 25 mm Tris ⁄ HCl, 140 mm NaCl,
pH 7.5. Detergent concentrations were chosen based on the
fact that Triton X-100 rafts are most often isolated using a
1% concentration, and the upper limit of the solubility of
Brij-96 at 4 °C is 0.5%. Each lysis buffer contained the
following protease ⁄ phosphatase inhibitors: 1 mm EDTA,
1mm phenylmethanesulfonyl fluoride and 1 mm Na

3
VO
4
,
plus a cocktail of protease inhibitors (Complete
TM
, EDTA-
free; Roche Diagnostics). Cells were left on ice for 20–
30 min to lyse, and then centrifuged at 10 000 g for 5 min
at 4 °C to obtain the postnuclear fraction. The postnuclear
lysate was adjusted to 40% (w ⁄ v) sucrose, and a 5–30%
(w ⁄ v) discontinuous sucrose gradient was layered on the
top. Typically, 2.2 mL of 30% (w ⁄ v) sucrose and 2.2 mL of
5% sucrose were layered on a 0.8 mL sample in 40%
sucrose in a 5.2 mL centrifuge tube. Samples were centri-
fuged (400 000 g,4°C, 3–4 h) in a VTi 65.2 rotor (Beck-
man Coulter, Mississauga, ON, Canada). Fractions of
0.4 mL (typically 12–13 fractions in total) were collected
from the top of the gradient using a Density Gradient
Fractionator (Brandel, Gaithersburg, MD, USA). Immuno-
blot analysis was performed to confirm which fractions
contained lipid raft microdomains.
Protein quantification
The bicinchoninic acid protein assay [49] was performed on
aliquots of the sucrose fractions, using BSA (crystallized
and lyophilized; Sigma) as a standard.
SDS/PAGE and western immunoblot analysis
Equal volumes of each fraction from the sucrose gradient
were analyzed by SDS ⁄ PAGE [50] on a 7.5% or 12% (w ⁄ v)
gel. Separated proteins were transferred to a nitrocellulose

membrane, which was blocked in 5% (w ⁄ v) milk in TBS-
Tween (0.05% (w ⁄ v) Tween-20 in 10 mm Tris, 100 mm
NaCl, pH 7.5), and then incubated with the primary anti-
body (C219, anti-caveolin-1, anti-Yes, anti-Fyn, anti-Lck or
anti-CD71). Membranes were subsequently incubated with
HRP-conjugated goat anti-mouse or goat anti-rabbit immu-
noglobulin, and developed using the Supersignal West PICO
enhanced chemiluminescence (ECL) system (Pierce Biotech-
nology Inc., Rockford, IL, USA).
Analysis of lipid raft-associated GM
1
ganglioside
Determination of lipid raft-associated GM
1
ganglioside was
carried out using a modification of the method of Blank
et al. [40], as described previously [37], using CTB–HRP
for detection and quantification. About 1–2 · 10
8
cells were
washed in ice-cold NaCl ⁄ P
i
, resuspended in 100 lL of ice-
cold NaCl ⁄ P
i
, and labelled with 600 mU of CTB–HRP for
30 min on ice. Cells were washed twice in ice-cold NaCl ⁄ P
i
(300 lL each wash) to remove unbound CTB–HRP, then
lysed in 300 lL of either 0.5% (w ⁄ v) Brij-96 or 1.0% (w ⁄ v)

Triton X-100 lysis buffer and subjected to sucrose density
centrifugation, as already described. A 50 lL aliquot of each
gradient fraction was assayed in a 96-well microplate, along
with a set of standards in the range of 0.002–1.0 mU of
CTB–HRP in the appropriate lysis buffer. To all standards
and samples, 50 lL of the chromogenic peroxidase
substrate, 2,2¢-E-azino-di-(3-ethylbenzthiazoline-6-sulphon-
ate) diammonium was added (Roche Diagnostics). After the
dark green color had developed, the absorbance of the sam-
ples was read at 405 nm using a plate-reader. GM
1
was also
analyzed by dot-blot analysis, in which an aliquot from each
fraction was spotted directly onto the nitrocellulose mem-
brane. GM
1
was visualized using CTB–HRP, followed by
ECL detection.
Cholesterol depletion
The procedure for cholesterol removal was adapted from
Sheets et al., with some modifications [51]. CH
R
B30 cells
were suspended in BSA-containing buffered saline solution
(BSA ⁄ BSS; 20 mm Hepes, 135 mm NaCl, 5 mm KCl,
1.8 mm CaCl
2
,1mm MgCl
2
, 5.6 mm glucose and

1mgÆmL
)1
BSA, pH 7.4) at a concentration of 6 · 10
7
cells
per mL. To deplete cholesterol, 20 mm MbCD was added
and cells were incubated for 30–60 min at 37 °C. Control
cells were incubated in BSA ⁄ BSS only. After treatment with
MbCD, cells were washed once in BSA ⁄ BSS to remove the
released cholesterol, washed once more in NaCl ⁄ P
i
, and
lysed in either Brij-96 or Triton X-100. Post-nuclear lysates
were recovered and separated on a sucrose density gradient.
Cholesterol analysis
Lipids were extracted from sucrose density fractions using
200–400 lL of CHCl
3
⁄ MeOH (2 : 1, v ⁄ v) for each 200–
400 lL fraction, and the lower organic phase containing the
lipids was transferred to a glass tube. Extracted lipids were
dried first under a stream of N
2
and subsequently in a
vacuum desiccator. The ferric chloride reagent was prepared
by diluting 2 mL of a stock solution of 2.5% (w ⁄ v) FeCl
3
in
H
3

PO
4
to 25 mL with concentrated H
2
SO
4
. To each dried
sample and a set of cholesterol standards, 0.75 mL of glacial
acetic acid and 0.5 mL of ferric chloride reagent were
added, mixing well with a vortexer after each addition [52].
After 5–10 min, a pink ⁄ purple color developed and the
absorbance of the samples was read at 562 nm.
Immunoprecipitation of Pgp and caveolin-1
Frozen CH
R
B30 cells were thawed and washed twice in
NaCl ⁄ P
i
. One millilitre of lysis buffer was added to a cell
P-glycoprotein in intermediate-density microdomains G. Radeva et al.
4934 FEBS Journal 272 (2005) 4924–4937 ª 2005 FEBS
pellet of 150–200 lL volume. The following lysis condi-
tions were tested: Brij-96 [0.5% (w ⁄ v) Brij-96 in 140 mm
NaCl, 25 mm Tris ⁄ HCl, pH 7.5]; Triton X-100 [1.0%
(w ⁄ v) Triton X-100 in 140 mm NaCl, 25 mm Tris ⁄ HCl,
pH 7.5]; Nonidet P-40 ⁄ Triton X-100 ⁄ octylglucoside [1.0%
(w ⁄ v) Triton X-100, 0.5% (w ⁄ v) Nonidet P-40, 60 mm
octylglucoside, in 140 mm NaCl, 25 mm Tris ⁄ HCl,
pH 7.5]. Cells were left on ice for 20–30 min to lyse and
then centrifuged at 10 000 g for 5 min at 4 ° C to obtain

the postnuclear fraction. Alternatively, after preclearing
the cell lysates of Brij-96 and Triton X-100 extracts, radio-
immunoprecipitation buffer was added to give final con-
centrations of 1% (w ⁄ v) Nonidet P-40, 0.5% (w ⁄ v) SDS,
in 140 mm NaCl, 25 mm Tris ⁄ HCl, pH 7.5. All lysis buff-
ers contained 2 mm EDTA, 1 mm phenylmethanesulfonyl
fluoride and a cocktail of protease inhibitors (Complete
TM
,
EDTA-free, Roche Diagnostics). Each lysate sample was
split into two 500 lL aliquots. One set of samples was
used for Pgp immunoprecipitation and the other for cave-
olin-1 immunoprecipitation. One microgram of anti-Pgp
or anti-caveolin-1 immunoglobulin was added and samples
were incubated for 2 h at 4 °C on a nutator. Immune
complexes were collected on Protein A–Sepharose beads
for 1 h at 4 °C on a nutator. Beads were then washed
twice in the appropriate buffer and the immunoprecipitat-
ed proteins were extracted in Laemmli sample buffer. One
half of each immunoprecipitation sample was run on
7.5% nonreducing SDS ⁄ PAGE (for Pgp western immuno-
blot analysis) and the other half was run on 12% non-
reducing SDS ⁄ PAGE (for caveolin-1 western immunoblot
analysis). Separated proteins were transferred to a nitrocel-
lulose membrane and analysed by western immunoblot
analysis and ECL detection.
Confocal fluorescence microscopy
The confocal fluorescence microscopy procedure was taken
from Roepstorff et al. with some modifications [53].
CH

R
B30 cells were grown on cover slips for 2–3 days to
subconfluent density. For coimmunolocalization of GM
1
ganglioside with Pgp and caveolin-1, GM
1
was labelled
prior to cell fixation. Cells were washed twice in NaCl ⁄ P
i
and incubated with cholera toxin B subunit (CTB) conju-
gated to Alexa 488 (5 lgÆmL
)1
; Molecular Probes, Eugene,
OR, USA) in a-MEM (no serum) with 0.2% (w ⁄ v) BSA,
for 1 h on ice. Unbound label was removed by three washes
in NaCl ⁄ P
i
. Cells were then fixed in 4% paraformaldehyde
in NaCl ⁄ P
i
for 20 min, and quenched with 1 m glycine for
2–5 min at room temperature. The plasma membrane was
permeabilized using 0.1% (v ⁄ v) Triton X-100 in NaCl ⁄ P
i
for 10 min and the cells were then washed twice in
NaCl ⁄ P
i
. Blocking was carried out overnight in 5% (w ⁄ v)
skim milk in NaCl ⁄ P
i

at 4 °C. Cells were incubated with
primary antibodies in NaCl ⁄ P
i
for 1.5 h (1 : 100 dilution)
and for 45 min with secondary antibodies in blocking
solution (1 : 100 dilution), with both incubations carried
out at room temperature. Secondary antibodies used were
goat anti-(mouse-Alexa
594
), goat anti-(rabbit-Alexa
594
) and
goat anti-(rabbit-Alexa
488
) (Molecular Probes). Cells were
washed twice in NaCl ⁄ P
i
, and cover slips were mounted
using Dako fluorescent mounting medium (Dako Corpora-
tion, Carpinteria, CA, USA). Fluorescence images were
obtained using a Leica DM-IRE2 inverted confocal micro-
scope (Leica, Wetzlar, Germany) and viewed with
63· lenses. Overlay images were analysed employing Leica
Confocal Software Lite.
Acknowledgements
We thank Dr Marc Coppolino, Department of
Molecular and Cellular Biology, for technical assist-
ance with the confocal microscopy, and Peihua Lu and
Joseph Chu for providing CH
R

B30 cells. This work
was supported by a Discovery Grant from the Natural
Sciences and Engineering Research Council of Canada,
and by a Research Grant from the Canadian Cancer
Society.
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