Caveolin-1 influences P2X
7
receptor expression and
localization in mouse lung alveolar epithelial cells
K. Barth
1,
*, K. Weinhold
1,
*, A. Guenther
1
, M. T. Young
2
, H. Schnittler
3
and M. Kasper
1
1 Institute of Anatomy, Medical Faculty ‘Carl Gustav Carus’, Dresden University of Technology, Germany
2 Faculty of Life Sciences, University of Manchester, UK
3 Institute of Physiology, Medical Faculty ‘Carl Gustav Carus’, Dresden University of Technology, Germany
Extracellular nucleotide signaling in vertebrate cells is
mediated by plasma membrane P2 receptors, which
may be divided into two categories: G-protein-coupled
receptors (P2Y) and ion channels (P2X) [1]. P2X
receptors are ATP-gated nonselective cation channels,
seven members of which (P2X
1)7
) have been cloned
and characterized in humans, rats and mice [2]. P2X
receptors display a unique topology among ion chan-
nels. The functional channel is a trimer, with each sub-
unit consisting of two transmembrane spans, a large

P2X7 receptor
Correspondence
M. Kasper, Institute of Anatomy, Medical
Faculty Carl Gustav Carus, Dresden
University of Technology, Fiedlerstr. 42,
D-01307 Dresden, Germany
Fax: +49 351 458 6303
Tel: +49 351 458 6080
E-mail:
*These authors contributed equally to this
work
(Received 3 January 2007, revised 11 April
2007, accepted 16 April 2007)
doi:10.1111/j.1742-4658.2007.05830.x
The P2X
7
receptor has recently been described as a marker for lung alveo-
lar epithelial type I cells. Here, we demonstrate both the expression of
P2X
7
protein and its partition into lipid rafts in the mouse lung alveolar
epithelial cell line E10. A significant degree of colocalization was observed
between P2X
7
and the raft marker protein Caveolin-1; also, P2X
7
protein
was associated with caveolae. A marked reduction in P2X
7
immunoreacti-

7
is not firmly embedded in rafts [14],
but may interact with an unknown raft component. In
this study, the authors imply that P2X
7
receptors in
nonraft microdomains of submandibular gland cells
are solely involved in ion channel activity, and that re-
ceptors present in lipid rafts may regulate the activities
of proteins included in signal transduction cascades
[14]. More evidence for the association of P2X
7
with
lipid rafts may be derived from the proteomic complex
described by Kim et al. [19]. In this study, several
potential interacting partners of rat P2X
7
were identi-
fied, including three chaperone proteins (Hsp70,
Hsp71, Hsp90) [19]. In addition, tyrosine phosphoryla-
tion of Hsp90 was implicated in the functional regula-
tion of ion channel activity [20]. Heat shock proteins
identified as part of the P2X
7
complex have been
shown to be localized to lipid rafts [21,22].
It is known that subsets of lipid rafts form cell
surface invaginations termed caveolae. Caveolae are
formed by polymerization of caveolins, hairpin-like
palmitoylated integral membrane proteins from lipid

7
protein expres-
sion are dependent upon Caveolin-1 expression in two
ways. First, we show that P2X
7
protein expression is
significantly reduced in Caveolin-1-knockout mice as
compared to wild-type mice. Second, we show that
short hairpin RNA (shRNA)-mediated downregulation
of Caveolin-1 expression leads to significantly reduced
levels of P2X
7
protein expression in E10 cells. These
data imply that Caveolin-1 regulates the expression
level of the P2X
7
receptor.
Results
Presence of the P2X
7
receptor and its
modification in alveolar epithelial cell lines
First, we tested the expression of the P2X
7
receptor in
permanent alveolar epithelial cell lines with properties
more similar to those of AT I cells (R3 ⁄ 1, L2, E10) or
AT II cells (A549) using western blot analysis. Ini-
tially, we were only able to detect P2X
7

7
(Fig. 1C). The 60 kDa band was unchanged
by N-glycosidase F treatment (Fig. 1C), and its mole-
cular mass was too low to represent nonglycosylated
full-length P2X
7
, raising the possibility that it might be
a nonspecific band. However, immunoreactivity to both
the 80 kDa and 60 kDa bands was abolished following
preincubation with the antibody control peptide
(Fig. 1D), implying that the 60 kDa band shared at
least a portion of the epitope recognized by the C-ter-
minal P2X
7
antibody. It is possible that the 60 kDa
band represents an alternatively spliced or N-terminally
truncated P2X
7
protein. However, because N-glycosyla-
tion is required for cell surface expression of functional
P2X receptors [6,7], it follows that the 60 kDa band, if
specific, represents a truncated, nonfunctional, intra-
cellular P2X
7
protein. We therefore assumed that
the 80 kDa band represents mature, functional P2X
7
Caveolin-1 and P2X
7
expresssion in lung cells K. Barth et al.

Another main characteristic of lipid rafts is low
buoyant density in sucrose gradient centrifugation. We
isolated raft-like membranes using three distinct meth-
ods; Triton X-100 treatment at 4 °C (Fig. 2B), a deter-
gent-free method (Fig. 3A), and Brij35 treatment at
4 °C (Fig. 3B). When rafts were prepared using Tri-
ton X-100 treatment followed by sucrose density gradi-
ent centrifugation, P2X
7
protein was not detected in
the Caveolin-1- and Flo-1-containing low-density raft
fractions (Fig. 2B, lanes 3–5), and was only faintly
detected in the nonraft fractions (Fig. 2B, lanes 9–13).
In contrast, significant amounts of P2X
7
protein were
detected in low-density raft fractions when rafts were
isolated using either a detergent-free raft isolation pro-
cedure (consisting of fine disruption of the membrane
by sonication followed by sucrose density gradient cen-
trifugation) (Fig. 3A, lanes 2–4) or Brij35 isolation
(Fig. 3B, lanes 3–4). P2X
7
protein was also detected in
the high-density fractions from both detergent-free
(Fig. 3A, lanes 7–9) and Brij35 isolation methods
(Fig. 3B, lanes 9–14), along with markers for Flo-1,
the Golgi apparatus and endoplasmic reticulum [b-coa-
tomer protein (b-Cop) and protein disulfide isomerase
(PDI)]. In addition, isolated lipid rafts prepared using

Nonglycosylated
Fig. 1. (A) Analysis of P2X
7
receptor expression in alveolar epithelial lung cell lines. P2X
7
receptor expression was measured in cell lysates
of A549, E10, R3 ⁄ 1 and L2 cells by SDS ⁄ PAGE and western blot analysis. Fifty micrograms of protein from each sample was loaded on the
gel. Western blot analysis was performed using rabbit anti-P2X
7
(1 : 375) and anti-c-tubulin (1 : 1000). c-Tubulin served as a loading control.
Representative data from three separate experiments are shown. (B) Immunoprecipitation of P2X
7
from R3 ⁄ 1 cells. The figure represents a
western blot for P2X
7
(1 : 1000) from samples immunoprecipitated with the same antibody (0.3 lg) to concentrate P2X
7
protein. Lane 1:
10 lg of rat P2X
7
from transiently transfected HEK cells. Note the appearance of a nonspecific 90 kDa band. Lane 2: total protein from
2.5 · 10
6
R3 ⁄ 1 cells shows a band of the same molecular mass as the positive control (lane 1; 80 kDa). (C) P2X
7
receptors are glycosylated
in E10 cells. Membranes were incubated in the presence of N-glycosidase F for 0.5 h, 1 h and 24 h, and compared with nontreated mem-
branes (–). Full deglycosylation of the 80 kDa band was indicated by a reduction in molecular mass of approximately 12 kDa to 68 kDa. The
mass of the 60 kDa band was unaffected by deglycosylation. Representative data from three separate experiments are shown. (D) Fifty
micrograms of E10 cell lysates were run on an SDS gel and blotted for P2X

7
at the plasma
membrane in this study (not shown); this was probably
due to poor preservation of the plasma membrane
under our experimental conditions rather than any
specific lack of receptor expression. When parallel
immunostaining of a similar area of E10 cells in a
second ultrathin section was performed, the majority
of caveolae were also positive with the polyclonal anti-
body to Caveolin-1–3 (Fig. 4, lower panel).
Double-label immunofluorescence of Caveolin-1
and P2X
7
in alveolar epithelial E10 cells
To examine the possible colocalization of the puriner-
gic P2X
7
receptor with Caveolin-1 in alveolar epithelial
E10 cells, we analyzed the immunocytochemical locali-
zation and distribution of both proteins with a laser
scanning confocal microscope, using two independent
channels (Fig. 5). Caveolin-1 and P2X
7
receptor are
endogenously expressed in E10 cells, and their localiza-
tion in subconfluent cells was determined. Figure 5A
shows a Caveolin-1 staining pattern at the cell surface,
confirming the known caveolar location of Caveolin-1.
In addition, intracellularly distinguishable punctate
patterns of Caveolin-1 staining were found (Fig. 5A).

lipid lipid rafts, and T1a as a marker of the nonraft fraction. Representative data from three separate experiments are shown. (B) Characteri-
zation of membrane fractions prepared by Triton X-100 from E10 cells. E10 cells were homogenized in a buffer containing 1% Triton X-100
and subjected to sucrose density gradient centrifugation. Thirteen fractions were collected (fraction 1, top of the gradient; fraction 13, bot-
tom of the gradient), and an aliquot of each fraction (20 lL) was resolved by SDS ⁄ PAGE and subjected to western blot analysis with anti-
bodies against Caveolin-1, P2X
7
, PDI, TfR, b-Cop and Flo-1. As expected, Caveolin-1 and Flo-1 were enriched in fractions 3–5, representing
caveolae-enriched membrane fractions. Representative data from three separate experiments are shown.
Caveolin-1 and P2X
7
expresssion in lung cells K. Barth et al.
3024 FEBS Journal 274 (2007) 3021–3033 ª 2007 The Authors Journal compilation ª 2007 FEBS
plasma membrane in a similar pattern as observed for
Caveolin-1 staining (Fig. 5B). Merged immunofluores-
cence pictures of Caveolin-1 and P2X
7
indicate partial
colocalization of both proteins (Fig. 5C). This partial
colocation implies that a portion of P2X
7
molecules is
located in or associated with lipid rafts of alveolar epi-
thelial E10 cells.
Caveolin-1-knockout animals show reduced P2X
7
expression levels
Immunohistochemical assessment of lungs from wild-
type and Caveolin-1-deficient mice revealed significant
differences in P2X
7

Cav-1
P2X
7
R
Flotillin-1
-Cop
PDI
TfR
A
B
Fig. 3. (A) Characterization of the membrane fractions prepared by sonication from E10 cells. Raft and nonraft membranes were prepared
by sonication followed by centrifugation in a discontinuous sucrose gradient. Nine fractions were collected (fraction 1, top of the gradient;
fraction 9, bottom of the gradient), and an aliquot of each fraction (20 lL) was resolved by SDS ⁄ PAGE and subjected to western blot analy-
sis with antibodies against Caveolin-1, P2X
7
, PDI, TfR, b-Cop and Flo-1. Representative data from three separate experiments are shown. (B)
Characterization of membrane fractions prepared by Brij35 from E10 cells. E10 cells were homogenized in a buffer containing 1% Brij35,
and subjected to sucrose density gradient centrifugation. Fourteen fractions were collected (fraction 1, top of the gradient; fraction 14, bot-
tom of the gradient), and an aliquot of each fraction (20 lL) was resolved by SDS ⁄ PAGE and subjected to western blot analysis with anti-
bodies against Caveolin-1, P2X
7
, PDI, TfR, b-Cop and Flo-1. As expected, Caveolin-1 and Flo-1 were enriched in fractions 3 and 4,
representing caveolae-enriched membrane fractions. Representative data from three separate experiments are shown.
Fig. 4. Immunogold detection of P2X
7
(upper panel) and Caveolin-
1–3 (lower panel) on an ultrathin cryosection of E10 cells. Note the
labeling of caveolae (black arrows). The thick arrow indicates a
caveosome. Bar ¼ 60 nm.
K. Barth et al. Caveolin-1 and P2X

7
of Caveolin-1 shRNA-treated E10 cells
revealed a partial loss of colocalization of P2X
7
, with
a remaining low amount of Caveolin-1 protein and an
altered intracellular distribution of P2X
7
(Fig. 7).
Discussion
The P2X
7
receptor was introduced as a novel marker
for alveolar epithelial type I cells [32], which cover
more than 95% of the alveolar surface in lungs. In our
study, we tested four alveolar epithelial cell lines with
characteristics of type I cells [33], and among them
only the E10 cell line expressed a robust amount of
the P2X
7
receptor. We determined a molecular mass
of 80 kDa of the P2X
7
receptor in E10 cells after
immunoblotting, consistent with the detected size of
A
Cav-1
B
P2X
7

4
receptor is required for its
localization to the cytoplasmic membrane. Glycosyla-
tion of the P2X
2
receptor is also required for their
functional surface expression [6,7].
In the present study, we further show that two
populations of the purinergic receptor P2X
7
are detect-
able in the plasma membrane of E10 cells. A discrete
population of P2X
7
was associated with lipid rafts, as
determined by cofractionation with the raft markers
Caveolin-1 and Flo-1 after preparation of lipid rafts in
the presence of Brij35 and detergent-free membrane
preparation. These fractions did not contain Golgi
apparatus and endoplasmic reticulum, as shown by
detection of the organelle-specific protein markers
b-Cop and PDI, respectively. A second population of
P2X
7
was present in higher-density membranes.
Interestingly, the majority of the P2X
7
receptor was
extracted by treatment with 1% ice-cold Triton X-100,
contrary to what would be expected for raft-embedded

7
R (80 kDa)
-tubulin
control shRNA1 shRNA2
0
20
40
60
80
100
120
percent (%)
P2X
7
R(80kDa)
Fig. 6. (A) Immunoperoxidase demonstration of P2X
7
in alveolar epithelial cells of wild-type (+ ⁄ +) and Caveolin-1-knockout (- ⁄ -) lungs. Wild-
type: Arrow indicates a negative alveolar epithelial type II cell and a neighboring positive type I cell. Note the additional strong immunoreac-
tivity of endothelial cells in a larger blood vessel (asterisk) and of capillary endothelial cells. Alveolar macrophages (arrowheads) were in most
cases negative. Knockout: Note the reduced immunoreactivity for P2X
7
in the entire lung parenchyma. Arrows indicate the loss of P2X
7
from
the alveolar lining layer. Bar ¼ 10 lm. (B) The effect of Caveolin-1 downregulation in E10 cells. After transfection of E10 cells with two dif-
ferent shRNA constructs of Caveolin-1 (shRNA1 and shRNA2) and three different scrambled shRNAs (control 1–3), cells were harvested,
and the expression levels of Caveolin-1 and of P2X
7
receptor were analyzed by western blot analysis with mouse monoclonal anti-Caveolin-1

interact weakly with an unknown component of the
rafts.
In Caveolin-1-knockout mice, both the formation of
caveolae and the expression of the P2X
7
receptor were
strongly reduced. This indicates that the Caveolin-1
protein itself or the formation of the caveolae may
directly affect the expression of the P2X
7
receptor. The
putative physiologic role of caveolae and Caveolin-1
could be to centralize, concentrate and colocalize part-
ner proteins, signaling proteins and effectors within
lipid rafts. For some proteins, Caveolin-1 may aid
localization of proteins to the plasma membrane
through a physical interaction. Brazer et al. [35]
showed that the transient receptor potential channel
protein (TRPC1) (amino acids 271–349) contains a
Caveolin-1-binding motif between amino acids 322 and
349. Deletion of this binding domain altered localiza-
tion of TRPC1 to the plasma membrane. Ion channels
are important components of many signal transduction
pathways; therefore, this localization may ensure that
the channels are located in proximity to the signaling
molecules that modulate them. Many signaling mole-
cules have been shown to be preferentially associated
with rafts [36].
To examine the possibility of a direct interaction
between Caveolin-1 and P2X

distribution in control and in Caveolin-1 shRNA-transfected E10 cells.
Note the loss of colocalization of Caveolin-1 and P2X
7
in Cav-1 shRNA treated cells.
Caveolin-1 and P2X
7
expresssion in lung cells K. Barth et al.
3028 FEBS Journal 274 (2007) 3021–3033 ª 2007 The Authors Journal compilation ª 2007 FEBS
The results presented in this study support the evi-
dence of active alveolar ion transport in alveolar
epithelial cells by adding P2X
7
, a potentially Caveo-
lin-1-regulated nonselective cation channel, to the list
of alveolar epithelial type I cell-specific channels [37].
The exact role of the P2X
7
receptor in alveolar liquid
homeostasis remains open. However, the P2X
7
recep-
tor not only gates the opening of cationic channels,
but also couples to various downstream signaling
events in the cell [10–14]. The P2X
7
receptor is also
thought to be able to mediate cell death by apoptosis
[38]; prolonged P2X
7
stimulation with extracellular

from BIOCHROM AG Seromed (Berlin, Germany).
Dimethylsulfoxide and loading buffer X were obtained
from AppliChem GmbH (Darmstadt, Germany). Eppend-
orf MasterMix was purchased from Eppendorf (Wesseling-
Berzdorf, Germany).
Cell culture
E10 cells were cultured in DMEM ⁄ Ham’s F12 medium
(1 : 1) supplemented with 10% fetal bovine serum and
2.5 mml-glutamine. They were grown at 37 °Cina5%
CO
2
atmosphere. Cells were seeded at a density of
3 · 10
4
cellsÆmL
)1
and passaged continuously. The culture
conditions for A549, L2 and R3 ⁄ 1 cells have been described
previously [25,40].
Triton X-100 solubility
Confluent cells of a T75 flask were washed twice with ice-
cold NaCl ⁄ P
i
(pH 7.2). Five hundred microliters of MBS
(25 mm Mes, pH 6.5, 150 mm NaCl) containing 1% Tri-
ton X-100 plus protease inhibitors was added to the cells.
After 30 min of incubation on ice, the soluble fraction was
collected. The remaining Triton X-100-insoluble fraction
was dissolved by adding 500 lL of 1% SDS to the T75
flask, and passed through a 26-gauge needle 10 times in

2
PO
4
, 0.1% Tween-20; pH 7.2–7.5) con-
taining 5% nonfat dry milk for 1 h at room temperature or
overnight at 4 °C, it was incubated with monoclonal mouse
anti-Caveolin-1 (clone 2297, dilution 1 : 1000 v/v; BD Bio-
sciences, Pharmingen, San Jose, CA, USA), polyclonal rab-
bit anti-P2X
7
(dilution 1 : 375 v/v; Sigma-Aldrich, Inc., St
Louis, MO, USA), monoclonal mouse anti-Flo-1 (clone 18,
dilution 1 : 1000 v/v; BD Biosciences), monoclonal mouse
anti-(human TfR) (clone H68.4, dilution 1 : 500 v/v; Zymed
Laboratories Inc., South San Francisco, CA, USA), poly-
clonal rabbit anti-PDI (dilution 1 : 750 v/v; StressGen Bio-
technologies Corp., Victoria, Canada), polyclonal rabbit
anti-b-Cop (dilution 1 : 750 v/v; Ongogene Research Prod-
ucts, Boston, MA, USA) and monoclonal mouse anti-
c-tubulin (clone GTU-88, dilution 1 : 1000 v/v; Sigma-
Aldrich) for 2 h at room temperature or overnight at 4 °C.
Next, the membrane was washed three times for 10 min.
Incubation with secondary horseradish peroxidase-conju-
gated antibodies [ECL anti-(mouse IgG), dilution
1 : 4000 v/v (Amersham Biosciences, Little Chalfont, UK),
or goat anti-(rabbit IgG), dilution 1 : 2000 v/v (Bio-Rad
K. Barth et al. Caveolin-1 and P2X
7
expresssion in lung cells
FEBS Journal 274 (2007) 3021–3033 ª 2007 The Authors Journal compilation ª 2007 FEBS 3029

using a 1 : 1000 v/v dilution of the
same antibody used in the immunoprecipitation.
Preparation of detergent-insoluble membrane
fractions
Preparation of detergent-insoluble membrane fractions was
carried out as described previously [42], with minor modifi-
cations. Confluent cells of six T75 flasks were washed twice
with ice-cold NaCl ⁄ P
i
(pH 7.2) and then scraped in 1 mL
of ice-cold MBS containing 1% Triton X-100 plus protease
inhibitors. After 30 min, the lysate was centrifuged at
2000 g for 10 min (Allegra
TM
64R, Beckman F2402H rotor;
Beckman Coulter, Fullerton, CA, USA). The supernatant
was removed from the pellet and stored on ice. The pellet
was resuspended in ice-cold MBS containing protease inhib-
itors, and homogenized by sonication (three times with 30 s
bursts). The homogenate was mixed with sucrose to a con-
centration of 40%, and 2 mL was placed at the bottom of
the centrifuge tube. Sucrose (35%, 1.4 mL) and 0.8 mL of
5% sucrose in MBS containing protease inhibitors were
layered on the top of the lysate. The gradient was centri-
fuged at 200 000 g for 20 h in an MLS 50 rotor (Beckman
Coulter). Fractions of 13 300 lL were collected, beginning
from the top of the tube. Experiments were performed at 4 °C.
For the preparation of Brij35-insoluble membranes, cells
were washed with ice-cold NaCl ⁄ P
i

(pH 11) plus
protease inhibitors. The cell suspension was homogenized
with a sonicator (three 30 s bursts). The homogenate was
mixed with sucrose to a concentration of 40%, and 2 mL
was placed at the bottom of the centrifuge tube. Sucrose
(35%, 1.4 mL) and 0.8 mL of 5% sucrose in MBS contain-
ing 500 mm Na
2
CO
3
(pH 11) and protease inhibitors were
layered on the top of the lysate. The gradient was centri-
fuged at 200 000 g for 20 h in an MLS 50 rotor (Beckman
Coulter). Five 300 lL fractions, followed by four fractions
of 600 lL, were collected, beginning from the top of the
tube. Experiments were performed at 4 ° C.
Deglycosylation
Deglycosylation was carried out using the N-glycosidase
F Deglycosylation Kit (Roche Diagnostics, Roche Applied
Science, Indianapolis, IN, USA). The concentration of the
samples was 25 lg of total protein in a volume of 5 l L.
We followed the protocol for complete deglycosylation
according to the manufacturer’s instructions. Five micro-
liters of reduced denaturation buffer was added to each
sample, with ensuing incubation at 95 °C for 3 min. The
samples were then mixed with 10 lL of reaction buffer and
10 lLofN-glycosidase F or 10 lL of reaction buffer (con-
trol). The incubation times were 30 min, 1 h and overnight
at 37 °C. For western blot analysis, the samples were mixed
with 6· SDS sample buffer.

incubated twice for 15 min with PBG (0.1 m NaCl ⁄ P
i
with
cold water fish skin gelatine and BSA-C; Aurion, Wagenin-
gen, The Netherlands). Washing in PBG was followed by
incubation with goat anti-(rabbit IgG), 12 nm gold (dilu-
tion 1 : 30 v/v) (Dianova, Hamburg, Germany). After six
additional 5 min washings in PBG, and a further six 5 min
washings in NaCl ⁄ P
i
, incubation with 2.5% (w ⁄ v) glutaral-
dehyde in NaCl ⁄ P
i
, and further brief washes in destilled
water, sections were stained by incubation with 2% methyl-
cellulose ⁄ 3% uranyl acetate (1 : 9 v/v) for 10 min.
Immunocytochemistry
Paraffin sections of mouse lung from wild-type (n ¼ 3) and
Caveolin-1-knockout mice (n ¼ 3) were used from a previous
study [44]. All immunocytochemical techniques have been
described previously [24]. Primary rabbit polyclonal antibody
was obtained from Sigma-Aldrich, Inc. (dilution 1 : 100 v/v)
and from Alomone Labs Ltd (Jerusalem, Israel) (dilution
1 : 1600 v/v). Double immmunofluorescence staining of E10
cells was performed by overlaying the acetone ⁄ methanol
(1 : 1 v/v) fixed cells with polyclonal rabbit anti-P2X
7
,dilu-
tion 1 : 25 v/v, for 30 min at room temperature. After exten-
sive washing with NaCl ⁄ P

nucleotides at the 5¢-end to generate a XhoI overhang. For-
ward and reverse oligonucleotides were annealed and
ligated into the Esp3I–XbaI site in the modified pLentilox
3.7 vector. The detailed sequences of the 19 nucleotide
sense strands of the shRNA
1
and shRNA
2
designed from
Caveolin-1 cDNA are GCAAGTGTACGACGCGCAC
and AACCAGAAGGGACACACA, respectively.
As negative controls (scrambled shRNAs), shRNAcontrol
1
(TAGCGACTAAACACATCAA), shRNAcontrol
2
(TATA
GCGACTAAACACATCAA) and shRNAcontrol
3
(AAAG
AGCGACTTTACACACTT) were designed. All constructs
were verified by sequencing.
shRNA-expressing lentiviruses were produced by triple
transfection of 293 T cells with Lentilox-Esp vector contain-
ing the shRNA (10 lg), and packaging vectors and pMD2G
carrying the vesicular stomatitis virus-glycoprotein, as des-
cribed elsewhere [46–48]. Twenty-four hours post-transfec-
tion, the lentiviruses containing supernatant were collected
and filtered through a 0.45 lm filter unit. The filtered solu-
tion was used directly to infect the E10 cell line. Viral stocks
were titrated by HIV-1 ELISA according to the manufac-

expresssion in lung cells
FEBS Journal 274 (2007) 3021–3033 ª 2007 The Authors Journal compilation ª 2007 FEBS 3031
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