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Centro Nacional de Biotecnología, Department of Immunology and Oncology, Campus de Cantoblanco, Universidad Autónoma de Madrid, Madrid, Spain
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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An important advance in understanding apoptosis has come from the description of the Bcl-2 gene family. Within this group, Bcl-x gene encodes several alternatively spliced protein isoforms that can enhance or diminish apoptosis after transfection (5, 6, 7, 8). The Bcl-xL inhibits cell death upon growth factor deprivation (9). A second Bcl-x isoform, Bcl-xS, encodes a smaller protein of 170 amino acids that enhances apoptosis (10). The murine Bcl-x gene family has been expanded to include two additional isoforms, which may inhibit apoptosis in B cells (7, 11). Bcl-xL contains a hydrophobic segment at the C-terminal end (12, 13, 14) that is believed to serve as a membrane anchor.
The Aiolos transcription factor has been identified as a homologue of the Ikaros transcription factor, whose expression is restricted to the lymphoid lineage. Aiolos homodimers are potent transcriptional activators, whereas the transcriptional activity of Aiolos-Ikaros heterodimers range from low to undetectable. Aiolos was first described in committed lymphoid progenitors and is strongly up-regulated as these progenitors become restricted to T and B lymphoid pathways (15). Aiolos plays an important role as a regulator of B cell differentiation, proliferation, and maturation to an effector state (16). The interplay between these proteins in the regulation of gene expression is further complicated by additional Ikaros isoforms or by other proteins that can sequester either Ikaros or Aiolos in transcriptional inner complexes (15). Aiolos is first detected at low level in double-negative thymocyte precursors and is up-regulated as these progress to double-positive stage. Aiolos expression decreases in splenic T cells. In thymus, Bcl-2 is expressed in double-negative cells and in a few double positives, as well as in nearly all single-positive cells and in mature T cells (17, 18, 19). These observations suggest that Aiolos and Bcl-2 expression occur in parallel throughout the lymphocyte differentiation stages. We have recently shown that IL-2 starvation induces Ras/Aiolos association, resulting in apoptotic cell death. One of the functional consequences of Ras/Aiolos interaction is the blockade of Aiolos translocation to the nucleus. In the absence of IL-2, dephosphorylated Aiolos is sequestered in the cytoplasm by Ras. IL-2 stimulation induces tyrosine phosphorylation of Aiolos and dissociation from Ras. We have identified functional Aiolos binding sites in the Bcl-2 promoter. Mutation of these Aiolos binding sites within the Bcl-2 promoter inhibits transactivation of the reporter gene, suggesting a direct control of Bcl-2 expression by Aiolos. Cotransfection experiments confirm that Aiolos induces Bcl-2 expression and prevents apoptosis in IL-2-deprived cells (20). In this study, we propose a model for the control of Bcl-xL anti-apoptotic role via association to the transcription factor Aiolos.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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TS1
is a murine T cell line stably transfected with the
human IL-2R - and -chains (21). This cell line
responds independently to IL-2, IL-4, or IL-9. Cells were cultured in
RPMI 1640 (BioWhittaker, Walkersville, MD), supplemented with 5%
heat-inactivated FCS (Life Technologies, Gaithersburg, MD), 2 mM
glutamine, 10 mM HEPES, 0.5 mM arginine, 0.24 mM asparagine, 50 µM
2-ME, and 60 U/ml IL-4.
Lymphokines, Abs, reagents, and plasmids
Murine rIL-4 or supernatant of a HeLa subline transfected with pKCRIL-4.neo was used as a source of murine IL-4. Anti-Bcl-xL Ab was from Calbiochem (La Jolla, CA) or Transduction Laboratories (Lexington, KY). Mouse pan-Ras was from Oncogene Research Products (Cambridge, MA). Anti-Aiolos polyclonal Ab was generated in our laboratory. Anti-PTyr Ab was from Transduction Laboratories. Anti-histone Ab was from Chemicon International (Temecula, CA). Peroxidase-conjugated goat anti-rabbit or anti-mouse Ig Ab was from DAKO (Glostrup, Denmark). ECL or ECL Plus were from Amersham (Amersham, U.K.). Nonidet P-40 was from Boehringer Mannheim (Mannheim, Germany). Propidium iodide (PI)2 and lysophosphatidylcholine were from Sigma-Aldrich (St. Louis, MO). Anti-Aiolos Ab was generated in our laboratory, and Cy3-, Cy2-, or Alexa 488-conjugated secondary Abs were from Molecular Probes (Eugene, OR). DEAE-Dextran was from Pharmacia (Uppsala, Sweden) and the Capture-Tec pHook3 kit was from Invitrogen (San Diego, CA). Expression vector pcDNA3-Bcl-xL was kindly provided by Dr. L. del Peso (Madrid, Spain). Expression vector pCIneo-Aiolos and production of specific anti-Aiolos Ab was previously described (20).
Transient transfection
TS1 cells were transiently transfected using the
DEAE-Dextran method. Cells (10 x 106) in
exponential growth were washed with TS buffer (25 mM Tris-HCl,
137 mM NaCl, 5 mM KCl, 0.7 mM CaCl2, 0.5 mM
MgCl2, 0.6 mM
Na2HPO4, pH 7.4). Cells
were transfected with pHook3 or pHook3 in combination with
Bcl-xL or with Bcl-xL and
Aiolos. 750 µl of TS buffer and 750 µl of freshly prepared
DEAE-Dextran (1 mg/ml) in TS buffer were mixed successively with the
cells and incubated for 20 min at room temperature, after which 13 ml
of RPMI 1640–5% FCS were added. Cells were incubated (1 h at 37°C),
centrifuged, and resuspended in 12 ml of RPMI 1640–5% FCS alone or
supplemented with 60 U/ml IL-4. The pHook3 vector drives the expression
of a hapten-specific single-chain Ab (sFv) on the surface of
transfected cells. Cells expressing the sFV were isolated from the
culture by binding to hapten-coated (pHox) magnetic beads and were
analyzed.
Estimation of apoptosis by PI staining
A total of 2 x 105 cells were washed and resuspended in PBS, permeabilized with 0.1% Nonidet P-40, and stained with 50 µg/ml PI immediately before analysis. Samples were analyzed using an Epics XL flow cytometer (Corixa, Miami, FL). Apoptosis was measured as the percentage of cells in the sub-G1 region of fluorescence scale having a hypodiploid DNA content.
Immunoprecipitation and Western blotting
Cells (1 x 107) were IL-4 stimulated or deprived for 12 or 24 h and lysed for 20 min at 4°C in lysis buffer (50 mM Tris-HCl, pH 7.5, 1% Nonidet P-40, 150 mM NaCl, 5 mM EDTA, protease and phosphatase inhibitor cocktail). Lysates were centrifuged (20 min, 15,000 rpm, 4°C) and either immunoprecipitated with the corresponding Ab (overnight at 4°C or for 2 h room temperature) or separated by SDS-PAGE. Protein A-Sepharose (20 µl) was added for 1 h at 4°C and, after six washing steps (50 mM Tris-HCl, pH 7.5, 1% NP40, 135 mM NaCla, 5 mM EDTA), immunoprecipitates were resuspended in Laemmli sample buffer and separated by SDS-PAGE. Alternatively, cells (5 x 105) were lysed in Laemmli sample buffer, and protein extracts were separated by SDS-PAGE, transferred to nitrocellulose, blocked with 5% nonfat milk for 2 h at room temperature in Tris-buffered saline (TBS, 20 mM Tris-HCl pH 7.5, 150 mM NaCl), and incubated with primary Ab (2 h at room temperature or overnight at 4°C) in TBS/0.5% nonfat dry milk. Membranes were washed with 0.05% Tween 20 in TBS and incubated with peroxidase-conjugated second Ab (1 h at room temperature). After washing (0.05% Tween 20/TBS), proteins were developed using the ECL system.
Two-hybrid screen
Bcl-xL cloned into the pGAD vector was
used as a bait to screen a cDNA library from TS1 cells in the
Saccharomyces cerevisiae L40 strain (MATa trp1
leu2 his3 LYS2::lexA-His3,
URA3::lexA-LacZ) using standard procedures.
Sequencing of inserts from positive clones of the two-hybrid screen was
performed on both strands with an automatic sequencer (model 373A,
Applied Biosystems, Foster City, CA). Sequences were compared using the
FASTA program.
Sense and antisense oligonucleotide
The phosphothioate analogs of the oligonucleotide from Aiolos, including the ATG initiation codon, were purchased from Isogen Bioscience. The sequence of the sense and antisense oligonucleotides are as follow: sense Aiolos, ATG GAA GAT ATA CAA; antisense Aiolos, TTG TAT ATC TTC CAT.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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cells
We have previously described that Bcl-2 is expressed in
IL-2-stimulated cells and Bcl-xL in IL-4-cultured
TS1 cells (22). We asked whether
Bcl-xL could replace the anti-apoptotic role
of Bcl-2 in IL-4-stimulated cells. When IL-4-maintained cells are
deprived of lymphokine, they undergo apoptosis (Fig. 1
A). As early as 4 h
after IL-4 deprivation, around 9% of cells were apoptotic, reaching
35%–40% at 24 h, whereas control IL-4-stimulated cells show no
apoptosis.
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cells were IL-4 stimulated or deprived for
different periods of time, and total Bcl-xL
expression was analyzed by Western blot. Bcl-xL
expression was observed in control IL-4-stimulated cells and was
similar throughout the deprivation period analyzed (Fig. 1
B). Although similar levels of Bcl-xL
were detected after IL-4 withdrawal, deprivation induced a strong
increase in apoptosis (Fig. 1A). We did not observe
expression of Bcl-xS (data not shown).
Because Bcl-xL does not promote cell survival in
the absence of IL-4, we investigated the mechanism by which
Bcl-xL was unable to inhibit apoptosis. Using
Bcl-xL as bait in the yeast two-hybrid system, we
screened a cDNA library from the TS1 cell. Among of the yeast
transformants screened, 41 clones were identified that interacted with
the Bcl-xL fusion protein. The clones were
sequenced. Seven of these were Aiolos, 24 were Bad, six were Bcl-G, and
four were Harakiri. The interaction between Aiolos and
Bcl-xL proteins is indicated by the induction of
LacZ expression (Fig. 2).
Neither Aiolos nor Bcl-xL alone restore
LacZ expression. We were not able to detect interaction
between Ras/Bcl-xL or Aiolos/Raf. Deletion of the
BH4 and BH3 domain of Bcl-xL abolishes
interaction with Aiolos (Fig. 2). Ras-Raf interaction was used as a
positive control. To analyze the cellular distribution of Aiolos,
total, nuclear, and cytoplasmic expression of Aiolos was studied in
IL-4 stimulated or deprived cells. When IL-4-maintained cells were
deprived of lymphokine, total Aiolos expression was not modified
throughout the starvation period analyzed, compared with control cells
(Fig. 3A). To study the
distribution of Aiolos, we performed Western blots of nuclear and
cytoplasmic extracts of IL-4-stimulated or -deprived cells. Similar
levels of Aiolos were detected in cytoplasmic extracts of
IL-4-stimulated or -deprived cells (Fig. 3B). As an internal
control of protein fractionation, hybridization with pan-Ras
(cytoplasmic marker) is shown, using nuclear proteins as a negative
control (Fig. 3B, lane N). As a control of
the proper protein fractionation procedure, membrane was proved with
anti-histone Ab. Similarly, Aiolos was detected in nuclear extracts
of IL-4-stimulated or -deprived TS1 cells (Fig. 3C),
suggesting that IL-4 deprivation does not induce trafficking of Aiolos
from the cytoplasm to the nucleus. Purity of protein fractionation was
detected by hybridization with anti-histone Ab (nuclear marker).
Cytoplasmic proteins were used as a negative control (Fig. 3C, lane C). The absence of nuclear
Bcl-xL expression was shown by probing the blot
with anti-Bcl-xL Ab.
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To analyze whether Bcl-xL and Aiolos could
interact in vivo, validating the results obtained in the two-hybrid
system, we performed reciprocal coimmunoprecipitation experiments of
cytoplasmic proteins under IL-4 stimulation or deprivation of TS1
cells. Bcl-xL was detected in anti-Aiolos
immunoprecipitates of IL-4-stimulated cells, increasing in 12-h and
24-h IL-4-deprived cells (Fig. 4A). Densitometric analysis of
Aiolos/Bcl-xL association shows an increase of
4-fold in IL-4-deprived cells compared with control IL-4-stimulated
cells. The specificity of this interaction was confirmed by
immunoprecipitation with an irrelevant Ab, anti-IL-2 (Fig. 4, A and B, lane I). Similar amounts
of Aiolos are shown in the cytoplasm of IL-4-stimulated or -deprived
cells (Fig. 4A). Purity of protein fractionation was
detected by hybridization with anti-histone Ab, using nuclear
extracts as a positive control (Fig. 4A, lane N).
In reciprocal experiments, Aiolos was detected in
anti-Bcl-xL immunoprecipitates of
IL-4-stimulated cells, increasing throughout the deprivation period
(Fig. 4B), showing an 3-fold higher association in
IL-4-deprived cells compared with control cells.
Bcl-xL protein does not change, as evidenced by
hybridization of the membrane with
anti-Bcl-xL Ab (Fig. 4B). The
specificity of the interaction was tested as above.
Aiolos/Bcl-xL interaction was also observed using
freshly isolated thymocytes, confirming the results obtained in vitro
(Fig. 4A, lane T). We were not able to detect
association of Aiolos and the survival molecule Bcl-3 (data not shown).
These results show that Aiolos interact with
Bcl-xL and that the association increases with
the deprivation period. SD of n = 7 is shown for Fig. 4, A and B. The t test for Fig. 4A shows p < 0.001 for 24-h samples and
p < 0.01 for 12-h samples. The t test for
Fig. 4B shows p < 0.0001 for 24-h samples
and p < 0.005 for 12-h samples. To estimate the total
Bcl-xL amount associated to Aiolos, supernatant
from Aiolos immunoprecipitates (10 x 106
cells) corresponding to 15% (Fig. 4C, lane 1,
1.5 x 106), 10% (lane 2,
1 x 106), 5% (lane 3,
0.5 x 106), 2.5% (lane
4, 0.25 x 106), and 1.25%
(lane 5, 0.12 x 106) of
total cells was transferred to nitrocellulose and blotted with
anti-Bcl-xL Ab. As shown in Fig. 4C, 10% of the total amount of
Bcl-xL was associated to Aiolos. It is
interesting to mention that we are estimating the association of
cytoplasmic Aiolos and Bcl-xL. We have analyzed
in detail by sucrose gradient the cellular distribution of
Bcl-xL in IL-4-stimulated cells showing that
65% of total Bcl-xL is associated to lipid
rafts and 35% is detected in cytoplasm (data not shown).
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To further analyze the mechanism by which
Bcl-xL and Aiolos interact in the cytoplasm of
IL-4-deprived cells, and given that IL-2 induces tyrosine
phosphorylation of Aiolos, we analyzed whether IL-4 could induce Aiolos
phosphorylation. Tyrosine-phosphorylated Aiolos was detected in Aiolos
immunoprecipitates of IL-4-stimulated cells, decreasing throughout the
deprivation period analyzed (Fig. 5A). Cytoplasmic
immunoprecipitated Aiolos was detected by reprobing the membrane with
anti-Aiolos Ab, showing similar levels. To verify that
Bcl-xL was associated to unphosphorylated Aiolos,
we performed Bcl-xL or Aiolos immunoprecipitation
of cytoplasmic proteins upon IL-4-stimulation or -deprivation
conditions. Tyrosine-phosphorylated Aiolos was detected in Aiolos
immunoprecipitates of control IL-4-stimulated cells (Fig 5B,
lane C). Tyrosine-phosphorylated Aiolos was not detected in
Bcl-xL immunoprecipitates of IL-4-stimulated or
-deprived cells (Fig. 5B). Immunoprecipitated Aiolos and
Bcl-xL were detected by reprobing the membrane
with anti-Aiolos and anti-Bcl-xL Ab.
Densitometric analysis of Western blot showed 3x more
Aiolos/Bcl-xL association in IL-4-deprived cells.
This result suggests that IL-4 induces tyrosine phosphorylation of
Aiolos, preventing its association with Bcl-xL
or, alternatively, that IL-4 may influence the state of
Bcl-xL and, consequently, its dissociation from
Aiolos.
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cells correlates with induction
of apoptosis without modification of Bcl-xL
expression and increased association of dephosphorylated Aiolos to
Bcl-xL, we hypothesized that
Bcl-xL would not be able to prevent apoptosis in
IL-4-deprived cells because this anti-apoptotic molecule is
associated to unphosphorylated Aiolos in the cytoplasm. Cells
transfected with Bcl-xL and deprived of IL-4 for
24 h show a strong reduction in the fraction of apoptotic cells
compared with IL-4-deprived mock-transfected cells (Fig. 6A). Transfected cells show
2-fold induction of Bcl-xL expression. To confirm
that Aiolos transcription factor associates
Bcl-xL, preventing its anti-apoptotic role,
cells were transfected with pHook3 or pHook3,
Bcl-xL, and Aiolos, and then were selected. Cells
transfected with Bcl-xL and Aiolos and deprived
of IL-4 for 24 h show a percentage of apoptosis comparable to that
observed in mock transfectants (Fig. 6A). The fraction of
apoptotic cells remains similar in control or transfected cells
maintained in IL-4. Expression of transiently transfected
Bcl-xL and Aiolos was confirmed by comparison of
Bcl-xL and Aiolos protein levels in transfected
cells and mock controls (Fig. 6B). Densitometric analysis of
Western blot of transfected cells shows an induction of
Bcl-xL and Aiolos expression in transfected
cells. To conclusively confirm that association of Aiolos to
Bcl-xL prevents its anti-apoptotic role, we
performed Bcl-xL immunoprecipitation in
Bcl-xL- or
Bcl-xL/Aiolos-transfected cells. Fig. 7 shows a high level of
Aiolos/Bcl-xL association in
Aiolos/Bcl-xL-transfected cells compared with
mock- or Bcl-xL-transfected cells, as observed
upon densitometric analysis of Western blot. Given that association of
Aiolos to Bcl-xL in IL-4-deprived cells blocks
the anti-apoptotic role of Bcl-xL, we
analyzed whether down-regulation of Aiolos expression in IL-4-deprived
cells would enhance cell survival by Bcl-xL. Fig. 8 shows that inhibition of Aiolos
expression by antisense oligonucleotide treatment enhances cell
survival by Bcl-xL in the absence of IL-4. The
inhibition of Aiolos expression upon antisense oligonucleotide
treatment was estimated by Western blot (Fig. 8). Taken together, these
results suggest that Bcl-xL is not able to act as
a survival factor in IL-4-deprived cells, because this
anti-apoptotic molecule is sequestered by Aiolos and only cells
overexpressing Bcl-xL are able to block apoptosis
in the absence of IL-4.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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cells by IL-4 induces Bcl-xL
expression, which was not altered throughout the starvation period
analyzed, suggesting that IL-4 deprivation-induced apoptosis proceeds
along pathways that do not involve changes in
Bcl-xL expression. It is also interesting to note
that IL-4-stimulated cells do not express the anti-apoptotic
molecule Bcl-2 (22).
It has been described in some cellular models that apoptosis induction
correlates with down-regulation of Bcl-2, but not Bcl-x, expression
(23, 24). Although Bcl-x can be up-regulated in T cells
and overexpression of Bcl-xL can enhance T cell
survival (11, 14, 25), physiologic expression of
Bcl-xL is not sufficient to confer resistance to
apoptosis after TCR ligation, because it is expressed equally well in
apoptotic and nonapoptotic T cell blasts, suggesting association with
other partners (26). The ability of
Bcl-xL to protect thymocytes from death signals
is counteracted by intracellular inhibitory proteins. There is
accumulating evidence that function of Bcl-xL is
modulated by several interacting proteins such as Bax, Bad, and Bag1
(12, 27, 28). This is also the case for TS1 cells
maintained in the presence or absence of IL-4. A significant proportion
of Bcl-xL is found to be cytosolic.
Hydropathic analysis of Bcl-xL sequence
predicts a transmembrane domain at the C-terminal region (9, 12). The observation that a significant fraction of
Bcl-xL is cytosolic suggests either that the
C-terminal hydrophobic domain may be hidden within the interior of the
protein or that Bcl-x may be involved in binding to other cytosolic
factors (29). These observations suggest that cytoplasmic
retention may be due to physical association with other proteins or to
posttranslational modifications.
Bcl-x can be alternatively spliced to produce two protein isoforms,
Bcl-xL and Bcl-xS (7, 9). IL-4-stimulated TS1 cells only express the
Bcl-xL isoform. Cell death by IL-2 deprivation
has been correlated, in some cellular models, with a decrease in the
level of Bcl-x (30), but in our experimental system,
Bcl-xL protein level was constant after IL-4
deprivation. Although Bcl-xL is expressed after
IL-4 stimulation, it seems to be insufficient to promote cell survival
because Bcl-xL is also expressed in IL-4-deprived
cells. A pathway different from Bcl-2 and Bcl-xL
may be triggered by IL-4 to prevent apoptosis. Alternatively,
Bcl-xL could not be able to function as an
anti-apoptotic molecule because it is physically associated with
other molecules in the cytoplasm of IL-4-deprived cells. IL-4
deprivation induces inhibition of Bcl-3 expression, resulting in
apoptotic cell death, which is blocked by Bcl-3 expression. This result
suggests that Bcl-3 can replace the anti-apoptotic role of Bcl-x,
acting as survival factor in IL-4-deprived TS1 cells. Bcl-3
down-regulation presumably results in a change in the regulation of
gene or genes important in some aspects of proliferation,
differentiation, or survival. Bcl-3 expression is probably controlled
by IL-4-regulated transcription factors through binding-site
recognition in the promoter region of Bcl-3. It has also found that
Bcl-3 is related to genes implicated in cell lineage determination and
cell cycle control (31). A correlation has also been
demonstrated between Bcl-3 expression and proliferation of B
lymphocytes (32). Taken together, these results suggest
transcriptional (Bcl-3 expression) (33) and
nontranscriptional (Bcl-xL/Aiolos interaction)
pathways involved in the control of apoptosis in IL-4-stimulated cells.
It is also interesting to note that Bcl-xL
interacts with Aiolos in freshly isolated thymocytes, confirming the
results obtained in vitro. In addition, we do not rule out that
Bcl-xL and Bcl-3 have a synergistic effect on the
control of apoptosis.
We have recently shown that IL-2 starvation induces association of Ras and dephosphorylated Aiolos (20), there is no Bcl-2 expression, and apoptosis is induced as a consequence. IL-4 stimulation does not induce Bcl-2 expression, probably due to the lack of Aiolos translocation to the nucleus, although Aiolos is expressed in the nucleus, suggesting that some induced factor(s) repress Bcl-2 transcription. Alternatively, low nuclear level of Aiolos is unable to induce Bcl-2 expression. IL-2 addition induces tyrosine phosphorylation of Aiolos and dissociation from Ras. Similarly, IL-4 induces tyrosine phosphorylation of Aiolos and dissociation from Bcl-xL. Our data demonstrate a specific interaction between Aiolos and Bcl-xL, and no Bcl-3, in IL-4-deprived cells as well as in thymocytes and provide an explanation for the inability of Bcl-xL to prevent apoptosis. In addition, we suggest that IL-4 plays an important role in the control of Aiolos/Bcl-xL interaction. The finding reported in this manuscript proposes a novel role for Aiolos in IL-4-deprived cells as a blocker of Bcl-xL anti-apoptotic function through its sequestering by unphosphorylated Aiolos. IL-4-induced tyrosine phosphorylation of Aiolos probably diminishes the affinity of Aiolos for Bcl-xL, inducing its dissociation. We do not exclude the possibility that phosphorylated Aiolos increases its affinity for other partners or that Aiolos/Bcl-xL interaction may also be regulated by other proteins. The threshold to apoptosis is controlled by a balance among survival proteins, their inhibitory partners, and the strength of death signals.
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Acknowledgments |
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Footnotes |
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2 Abbreviation used in this paper: PI, propidium iodide.
Received for publication May 29, 2001. Accepted for publication September 24, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-4 deprivation; 
,
IL-4 stimulation. B, Expression of the transiently
transfected Bcl-xL and Aiolos was confirmed by direct
comparison of Bcl-xL and Aiolos protein levels in mock and
transfected cells. The blot was probed with pan-Ras as internal control
of protein loading. Similar results were obtained in two independent
experiments. Densitometric analysis of Bcl-xL and Aiolos
Western blot is shown.
