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Division of Reproductive Biology (S.Y.H., A.K., A.J.W.H.)
Department of Gynecology and Obstetrics Stanford University Medical
School Stanford, California 94305-5317
Division of
Molecular Biology Application (L.Z.) CLONTECH Lab, Inc. Palo
Alto, California 94303-4607
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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BAD is a new member of the Bcl-2 family; it shares the conserved Bcl-2 homology (BH)1 and BH2 domains with other Bcl-2 family members and counters the antiapoptotic effects of Bcl-xL in an interleukin 3-dependent cell line (7). However, BAD does not have the C-terminal transmembrane domain found in most Bcl-2 family members but contains unique hydrophilic PEST motifs (7) postulated to be targets of protease degradation (8). Based on the unique structural features of BAD, we hypothesized that BAD may regulate apoptosis in a manner different from other Bcl-2 family members and interact with cellular proteins outside the Bcl-2 family.
In a recent report using expression cloning of an embryonic cDNA library, the interaction between mouse BAD and 14–3-3 proteins was demonstrated (9). In addition, serine phosphorylation of BAD was shown to be important for 14–3-3 binding and cell survival. We used the yeast two-hybrid system to search for BAD-interacting proteins and also found that different isoforms of the 14–3-3 protein bind strongly to BAD through a putative 14–3-3 binding site. The highly conserved ubiquitous 14–3-3 family of proteins is expressed in diverse eukaryotic organisms (10). They are capable of binding to a variety of protooncogenes and key enzymes important in different intracellular signaling pathways including mitogen-activated cell cycle progression, signal transduction mediated by protein kinase C isoforms, and oncogenesis (11, 12, 13, 14, 15, 16, 17). We found that overexpression of 14–3-3 suppresses apoptosis induced by BAD in Chinese hamster ovary (CHO) cells, indicating that interactions between them may allow coordination of cell death and 14–3-3-regulated intracellular signaling pathways. Using a BAD mutant (S137A BAD) not capable of binding 14–3-3 as a bait in the yeast two-hybrid system, we further identified another BAD-interacting protein P11. P11, also known as 42C or calpactin I light chain, is an early response gene induced by nerve growth factor (NGF) and found to be essential for neuronal cell survival and neurite formation (18, 19). Overexpression of P11 was also found to partially block BAD-induced apoptosis in CHO cells.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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, and 
forms of 14–3-3 cDNAs.
Sequence analysis also showed that one cDNA clone for the ß-form of
14–3-3 encodes a novel splicing variant with a shorter 3'-
untranslated region. Studies using yeast cells further indicated
comparable interactions between BAD and four 14–3-3 isoforms (ß,
, , and 
; Fig. 1A
; left
panel). In the same assay, neither Bcl-2 (Fig. 1A; right
panel) nor Bax or lamin C (data not shown) interacted with any of
the 14–3-3 proteins, suggesting the interaction between BAD and
14–3-3 is specific. Independent verification of protein-protein
interactions was confirmed based on activation of the GAL4-lacZ
reporter expression (data not shown).
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and Ref.16). To test the structural requirement for
interactions between BAD and 14–3-3, BAD mutants with single amino
acid changes within the putative 14–3-3 binding motifs or with
truncations were generated (Fig. 1B and Ref.22). In yeast two-hybrid
analysis (Fig. 1C), S137A, but not S113A, mutation in the binding motif
of BAD abolished interaction of BAD with 14–3-3. Likewise, double
mutations in both sites also abolished BAD binding to 14–3-3. The
effect of the S137A mutation is context-specific because this mutant,
like the wild type protein, still interacted with Bcl-2. Furthermore,
truncation of residues 1–140, but not 1–42, 1–61, or 189–205, in
BAD disrupted its interaction with 14–3-3 (Fig. 1C).
Identification of P11 as a Binding Protein for Underphosphorylated
BAD: Preferential Binding of P11 to the S137A BAD Mutant
Phosphorylation of the second serine in the 14–3-3 binding
site is essential for interaction of 14–3-3 with different signaling
proteins (16). We hypothesized that the S137A BAD mutant not capable of
binding 14–3-3 resembles an underphosphorylated form of BAD and used
it as a bait to screen for an additional 1.5 million yeast
transformants expressing rat ovarian fusion cDNAs. Among 13
positive clones, eight encoded P11, also known as 42C or calpactin I
light chain. As shown in Fig. 2A
(left panel), yeast cells coexpressing P11 and the S137A BAD
mutant showed pronounced growth whereas minimal growth was found in
cells coexpressing P11 and the wild type BAD. Likewise, the S113A/S137A
BAD double mutant not capable of binding 14–3-3 also showed strong
interaction with P11. In the same assay, wild type BAD, but not its
S137A and S113A/S137A mutants, interacted with 14–3-3 (Fig. 2A;
right panel). Independent verification of protein-protein
interactions between S137A BAD and P11 was confirmed based on
activation of the GAL4-lacZ reporter expression. Using the same assay,
no interaction of P11 with Bcl-2, BAX, or lamin C was observed (data
not shown). These data suggested that underphosphorylated S137A BAD
binds preferentially to P11.
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).
Induction of Cell Death after Overexpression of Wild Type and
Mutant BAD in CHO Cells and Blockage by Baculovirus Apoptosis Inhibitor
P35
To investigate the role of BAD and its interacting proteins on
cell survival, a ß-galactosidase cotransfection assay was used to
examine BAD activity (3, 23). CHO cells were transfected with various
expression vectors together with a 1/10 equivalent of the pCMV-ß-gal
plasmid. After 36 h, cells were stained with X-gal to identify
transfected blue cells for examination of morphological signs of cell
death. As shown in Fig. 3A (left
panel), cells transfected with an empty pcDNA3 vector showed
normal spindle-shaped morphology with minimal cell death. In contrast,
most cells transfected with the BAD expression plasmid showed
characteristics of apoptosis including cell shrinkage, a round-up
shape, and cytoplasmic fragmentation (Fig. 3A; right panel).
Immunocytochemical analysis (Fig. 3B) showed BAD expression in both
normal and apoptotic cells transfected with the BAD expression vector
but not in cells transfected with the empty vector. Quantification of
cell death using the cotransfection assay indicated that >55% of
cells underwent apoptosis after BAD expression. Similar to wild type
BAD, overexpression of the S113A and S137A BAD mutants also increased
the percentage of cells undergoing apoptosis (P <
0.01) whereas cells transfected with a plasmid with BAD in reverse
orientation did not (Fig. 3C). Western blot analysis further
demonstrated that wild type BAD migrated as two bands whereas only the
lower band was seen for cells expressing the mutants (Fig. 3C, lower panel).
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, induction of apoptosis by
BAD or its S137A and S113A mutants was reduced after coexpression with
P35 (P < 0.01), suggesting the involvement of a
caspase-mediated proteolysis cascade. In contrast, transfection with
the P35 expression vector alone did not affect cell survival.
Suppression of BAD-Induced Apoptosis by 14–3-3 and P11 in CHO
Cells
To test the ability of 14–3-3 to modulate BAD-induced apoptosis,
CHO cells were cotransfected with vectors encoding BAD and the
ß-isoform of 14–3-3 (Fig. 4A). The
ability of wild type or S113A BAD to induce apoptosis was reduced after
cotransfection with an equivalent amount of the 14–3-3 expression
vector (P < 0.01). In contrast, apoptosis induced by
the S137A BAD mutant was not affected by 14–3-3 coexpression,
consistent with its inability to bind 14–3-3. Also, transfection with
the 14–3-3 expression vector alone did not affect cell survival.
Western blot analysis of cell lysate showed the expression of BAD
proteins with apparent molecular masses of 27 and 28 kDa in cells
transfected with the expression plasmid encoding wild type BAD, while
the mutant proteins migrated as a single band of 27 kDa (Fig. 4A, lower panel). The higher molecular mass band presumably
represents phosphorylated BAD. Of importance, the BAD antigen level did
not decrease in cells coexpressing BAD and 14–3-3, suggesting that the
observed attenuation of BAD action by 14–3-3 is not due to changes in
BAD expression. In addition, Western blot analysis using a
ß-isoform-specific 14–3-3 antibody indicated that cotransfection
with the 14–3-3 expression vector increased the level of this protein
in transfected cells (Fig. 4A, bottom panel).
Immunoprecipitation analysis further confirmed direct interaction
between BAD and 14–3-3 in CHO cells. Incubation of lysate of cells
expressing wild type BAD, but not lysates from cells transfected with
the empty vector, with an anti-BAD antibody resulted in the
precipitation of 14–3-3 proteins (Fig. 4B).
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). Although
overexpression of P11 alone did not affect the percentage of cells
undergoing apoptosis, coexpression of P11 decreased apoptosis induced
by either wild type BAD or the S137A BAD (P < 0.01).
Furthermore, Western blot analysis indicated that cotransfection with
P11 did not affect the amount of BAD antigen expressed (Fig. 5A, lower panel), suggesting the blockage of BAD action by P11
was not due to uneven protein expression. To further substantiate the
interaction between P11 and BAD observed in yeast cells, a glutathione
S-transferase (GST) fusion protein system was used.
Bacteria-derived recombinant GST-BAD, a fusion protein of BAD and GST,
was immobilized onto glutathione-Sepharose beads and used to confirm
the interaction between P11 and BAD (Fig. 5B). Recombinant P11 protein
tagged with a hemagglutinin epitope (HA-P11) bound to beads containing
the GST-BAD fusion protein but did not bind to beads containing GST
alone, further suggesting that P11 directly interacts with BAD without
posttranslational modification.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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BAD was originally isolated as a Bcl-2-binding protein that blocks the antiapoptotic effect of Bcl-xL in an interleukin 3-dependent cell line (7). Because BAD interacts only with the antiapoptotic proteins Bcl-xL and Bcl-2 and does not promote cell death by itself in the interleukin 3-dependent cell line, BAD was proposed to promote apoptosis by dimerization with Bcl-xL and displace the apoptosis promoter Bax (7). In this study, transient overexpression of BAD alone in CHO cells induces apoptosis, thus providing a convenient system to study the role of BAD and its binding proteins in modulating apoptosis. Because the baculovirus serpin inhibitor P35 blocked BAD-induced apoptosis, the pro-apoptotic action of BAD may involve a caspase-mediated proteolysis cascade. Dimerization among different Bcl-2 family members is mediated by the conserved BH1 and BH2 domains (26, 27). Although the exact Bcl-2 family member serving as the dimerization partner for BAD in CHO cells is unclear, BAD could exert its pro-apoptotic action by blocking the antiapoptotic effect of one of the survival genes in the Bcl-2 family. A fundamental question in apoptosis research is the mechanisms by which hormonal and other extracellular survival signals regulate the Bcl-2 and the downstream caspases. Identification of 14–3-3 and P11 as binding proteins for BAD and the observation that 14–3-3 and P11 both attenuate apoptosis induced by BAD suggest that the pro-apoptotic activity of BAD could be modulated by multiple signaling proteins outside the Bcl-2 family.
The highly conserved ubiquitous 14–3-3 proteins are expressed in perhaps all eukaryotic organisms, including yeast, plants, insects, and mammals, with at least seven different isoforms identified in mammalian cells (10). Proteins of the 14–3-3 family bind diverse enzymes and signaling molecules, including Raf-1 kinase, B-Raf, phosphatidyl inositol 3 kinase, CDC25 phosphatases, Bcr, Cbl, and polyoma middle T antigen (14). They are important in intracellular signaling, cell cycle control, oncogenesis, and neurotransmitter biosynthesis in neuron (10). Crystallographic studies revealed that different isoforms of 14–3-3 dimerize to generate a complex with two ligand-binding sites (28, 29), thus allowing the assembly or anchoring of functional complexes containing diverse signaling proteins and cytoskeletal elements (15, 16, 17). Although the mechanisms by which 14–3-3 interferes with BAD-induced apoptosis remain to be defined, it is possible that 14–3-3 proteins may bring BAD to the proximity of specific enzymes, allowing cross-talks between BAD and different signaling pathways. Alternatively, this interaction may serve to prevent BAD from interacting with downstream effectors. After treatment with interleukin-3, FL5.12 cells showed an increase in BAD phosphorylation. Subsequent binding of 14–3-3 to phosphorylated BAD prevents BAD interaction with the membrane-bound BclxL, thus freeing BclxL to function as a survival protein (9). Our findings are consistent with these studies and further suggest that overexpression of 14–3-3 could suppress BAD-induced apoptosis. Because 14–3-3 proteins bind and modulate the activity of Raf-1 (30, 31, 32, 33), interactions between 14–3-3 and BAD may allow coordinated regulation of cell cycle progression and apoptosis.
Phosphorylation of the second serine in the 14–3-3 consensus-binding
site (RxRSxSxP) is essential for interaction between 14–3-3 and its
binding proteins (14, 16). Among the two potential 14–3-3-binding
sites in BAD (Fig. 1B), the second shows complete consensus whereas the
first is less conserved. In our study, mutation of serine 137 in the
second consensus site, but not serine 113 in the first site, abolished
BAD binding in yeast cells. Likewise, overexpression of 14–3-3 in CHO
cells attenuated apoptosis induced by wild type BAD or its S113A
mutant, whereas the S137A mutant that is incapable of binding 14–3-3
in yeast cells retained its apoptosis-inducing activity even when
14–3-3 was coexpressed. Thus, wild type BAD is presumably
phosphorylated at serine 137 whereas the S137A mutant resembles an
underphosphorylated form and shows minimal interaction with 14–3-3. In
contrast to our data using both yeast and CHO cells, S137A BAD mutant
could be coprecipitated with 14–3-3 in FL5.12 cells (9), thus
suggesting the phosphorylation pattern of BAD is probably cell
type-specific, as determined by the levels of different kinases. Our
finding that the S137A BAD mutant could not induce apoptosis more than
wild type BAD further suggests that endogenous 14–3-3 proteins may be
compartmentalized in CHO cells.
P11 is also known as 42C or calpactin I light chain and belongs to the S100 family of calcium-binding proteins (18). It was originally identified as an early response gene after NGF stimulation of a rat pheochromocytoma (PC12) cell line (18). Of interest, overexpression of P11 induces neurite outgrowth and enhances PC12 cell survival in the absence of NGF (19). This protein exists in an unstable soluble form by itself or complexes with P36 to form a stable and membrane-bound calpactin I tetramer of (P11)2 (P36)2 (34). In diverse cell lines, both P11 and P36 were increased after transformation induced by viral oncogenes (35). The present finding of P11 as a BAD-interacting protein capable of attenuating the pro-apoptotic effect of BAD provides a molecular mechanism for the observed survival function of P11. However, P11 appears to be less effective than 14–3-3 in the present transfection assay, probably because it is a labile protein. Because cellular levels of P11 are stabilized by P36 or annexin II (34), initially identified as a substrate for the transforming protein of Rous sarcoma virus (36), it is of interest to study the effect of coexpressing P11 and P36 on BAD regulation of apoptosis. PCTAIRE-1, a protein homologous to cyclin-dependent kinases, was also found to interact with both 14–3-3 and P11 (37). Although binding domains between specific protein pairs have not been characterized, it is likely that 14–3-3 and P11 could interact with different proteins through similar motifs, and P11 could have a role in other 14–3-3-regulated signaling pathways in addition to apoptosis regulation. Since the expression of P11 is hormonally regulated and tissue-specific (18), the present observation indicated that extracellular hormonal signals could modulate BAD-induced apoptosis through induction of BAD-binding proteins in addition to regulating the interaction between BAD and 14–3-3 via BAD phosphorylation (9). Future studies will reveal the role of P11 and BAD in hormonally regulated apoptosis models such as ovarian follicles, from which these cDNAs were isolated.
The use of the S137A BAD mutant with one less hydroxyl group as a bait
allowed us to isolate P11, which showed minimal binding with the
presumably phosphorylated wild type BAD in yeast. However, coexpression
of P11 attenuated the pro-apoptotic activity of both wild type and
S137A BAD in CHO cells. Because immunoblot analysis suggests BAD in CHO
cells existed in both phosphorylated and underphosphorylated forms and
recombinant P11 could interact with wild type BAD in vitro,
P11 could prevent BAD-induced apoptosis by interacting preferentially
with the presumably pro-apoptotic, underphosphorylated form of BAD in
CHO cells. These data suggest the phosphorylation status of BAD is
critical for the regulation of its proapoptotic activity;
phosphorylated BAD interacts with 14–3-3 whereas underphosphorylated
BAD preferentially interacts with P11 (Fig. 6). Identification of 14–3-3 and P11 as
BAD-interacting proteins expands recent findings showing Bcl-2
interaction with other non-Bcl-2 family proteins, such as BAG-1, Nip1,
Nip2, Nip3, Raf-1, R-ras p23, 53BP2, and Ha-Ras p21 (38, 39, 40, 41, 42, 43).
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). These control mechanisms
may serve as key steps to integrate various hormonal signal
transduction pathways and the apoptosis machinery, thus maintaining
homeostasis. |
MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Interactions between BAD and its binding proteins were assessed further using the pGBT9 GAL4-BD and pGADGH GAL4-AD vectors (21). Specific binding of different protein pairs in yeast was evaluated based on the activation of GAL1-HIS3 and GAL4-lacZ reporter genes. A minimum of three independent transformants with each pair of hybrid cDNAs were analyzed for the expression of two reporter genes. For GAL1-HIS3 reporter expression, cells were grown in a medium lacking leucine, tryptophan, and histidine but contained 30 mM 3-aminotriazole to inhibit endogenous histidine production. The activation of the GAL4-lacZ reporter gene was monitored using a filter lift assay for ß-galactosidase. Yeast cells patched on leucine(-) and tryptophan(-) plates were incubated for 36 h at 30 C before lysis by freezing and thawing and placed on filters presoaked in Z buffer (Na2HPO4, 10 mM KCl, 1 mM 2-mercaptoethanol, pH 7.0) containing 0.4 mg/ml 5-bromo-4-chloro-3-indolyl-ß-D-galactoside. Appearance of a blue color indicated ß-galactosidase activity.
Construction of Expression Vectors Encoding BAD Mutants
The serine to alanine BAD mutants were generated using
oligonucleotide-directed, two-step PCR mutagenesis (22), whereas the
truncated BAD mutants were derived using PCR amplification. For yeast
studies, mutant cDNAs were subcloned into the pGBT9 expression vector,
whereas the same cDNAs were subcloned into the pcDNA3 expression vector
(Invitrogen, Inc., San Diego, CA) for mammalian cell studies.
Analysis of Apoptosis in Transfected CHO Cells
Apoptosis was monitored after transfection of different cDNAs as
previously described (3, 23). CHO cells were plated at a density of
2 x 105 cells per well in DMEM/F12 supplemented with
10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2
mM glutamine. One day later, cells were transfected using
the lipofectamine procedure (Life Technologies, Gaithersburg, MD) with
the empty pcDNA3 expression vector or the same vector containing
different cDNAs, together with 1/10 equivalent of an indicator plasmid
pCMV-ß-gal to allow the identification of transfected cells.
Inclusion of 10-fold excess expression vectors, as compared with the
pCMV-ß-gal reporter plasmid, ensured that most of the
ß-galactosidase-expressing cells also expressed the protein(s) under
investigation. Cells were incubated with plasmids in a serum-free
medium for 4 h, followed by the addition of FBS to a final
concentration of 5% and further incubation for 14 h. After an
additional culture in fresh medium for 18 h, cells were fixed by
0.25% glutaraldehyde and stained with X-gal [0.4 mg/ml in buffer
containing 150 mM NaCl, 100 mM
Na2HPO4, 1 mM MgCl2,
3.3 mM K4Fe(CN)6. 3H2O,
and 3.3 mM K3Fe(CN)6, pH 7.0] for
6 h at 37 C to detect ß-galactosidase expression. The number of
blue cells were counted by microscopic examination and scored as either
live (flat or spindle-shaped blue cells) or dead (fragmented or
rounded-up blue cells) (3, 23). Data are expressed as the percentage
(mean ± SEM) of blue cells exhibiting signs of
apoptosis based on counting of six independent samples (at least 500
cells per 35-mm dish) from three or more separate experiments.
Statistical differences among treatment groups were analyzed using
one-way ANOVA and Scheffe F-test.
Immunoblotting and Immunocytochemical Studies
At 36 h posttransfection, CHO cells were washed with PBS
and lysed in NP-40 lysis buffer (100 mM NaCl, 20
mM Tris, pH 8.0, 1 mM EDTA, and 0.1% NP-40)
supplemented with 1 mM phenylmethylsulfonyl fluoride and 1
µg/ml leupeptin. Protein concentrations were determined by the
Bradford method (Bio-Rad Laboratory, Hercules, CA). Aliquots of cell
lysates were then boiled in Laemmli solubilization buffer before being
electrophoresed on 13–15% SDS polyacrylamide gels, transferred onto
Immobilon-P membranes, and blotted for 2 h with primary antibodies
(at 1:5,000 for BAD and at 1:20,000 for ß 14–3-3; Santa Cruz
Biotechnology, Santa Cruz, CA). After washing with Tris-buffered saline
(TBS)/0.1% Tween 20, membranes were incubated with a goat anti-rabbit
second antibody conjugated to horseradish peroxidase, and signals were
detected using enhanced chemiluminescence (ECL) (Amersham, Arlington
Heights, IL). For immunocytochemistry of BAD, cells were fixed and
quenched with 1% hydrogen peroxide in methanol for 30 min, followed by
three washes in TBS. After incubation with 5% nonimmune goat serum in
TBS, cells were treated with a polyclonal antibody against BAD (at
1:200 in TBS; Santa Cruz Biotechnology) for 30 min, followed by TBS
washing (8 x 3 min) before incubation (30 min) with a goat
anti-rabbit second antibody conjugated to horseradish peroxidase
(Amersham). The signals were developed using the substrate
3,3-diaminobenzidine (Vector Laboratories, Burlingame, CA).
Coprecipitation Experiments
To confirm interactions between BAD and 14–3-3, CHO cells
transfected with the expression vector encoding BAD or an empty vector
were harvested at 36 h after transfection. After washing, cells
were lysed in binding buffer (50 mM Tris, pH 7.5, 150
mM NaCl, 10 µg/ml trypsin inhibitor, and 0.1% NP-40),
and the supernatant was precleared with 1 µg/ml of nonimmune goat
serum and 10 µl Protein A Sepharose (Pharmacia Biotech, Uppsala,
Sweden). After removal of Protein A Sepharose beads by centrifugation,
cell lysates were incubated with the anti-BAD antibody for 3 h and
Protein A Sepharose (50 µl) for 1 h at 4 C, washed three times
with the binding buffer, and boiled in a 4x Laemmli buffer for 5 min.
Immunoprecipitated proteins were electrophoresed using 13% SDS-PAGE
and analyzed for the presence of 14–3-3 with an anti-14–3-3 antibody
(K19 at 1:5,000, Santa Cruz Biotechnology).
In Vitro Protein Interaction Assay
Recombinant hemmagglutinin epitope-tagged P11 (HA-P11) and
GST-BAD were prepared by subcloning rat P11 and BAD cDNAs into
prokaryotic expression vector pTricB and pGEXT-4T-1 (Invitrogen),
respectively. Plasmids were transformed into DH5-á bacteria, and
protein synthesis was induced with 0.5 mM
isopropyl-ß-D-thiogalactoside. The bacteria were lysed in
PBS containing 1% Triton X-100, sonicated, and clarified by
centrifugation before purification of recombinant proteins using
Nickle-NTA-agarose (Qiagen Inc., Chatsworth, CA) or GST-Sepharose
(Pharmacia Biotech) chromatography. In vitro binding assays
were performed by incubating equal amounts of recombinant GST or
GST-BAD immobilized onto glutathione-Sepharose beads, with recombinant
HA-P11 diluted in 0.4 ml Tris buffer (20 mM, pH 8.0)
containing 100 mM NaCl, 0.15% NP-40. The slurry was
incubated at 4 C for 4 h, washed five times with the same buffer,
and resuspended in Laemmli buffer. Proteins retained on the agarose
beads were boiled for 5 min and resolved using 15% SDS-PAGE before
analysis using an anti-HA-peroxidase monoclonal antibody (Boehringer
Mannheim, Indianapolis, IN) or a goat anti-GST polyclonal antibody
(Pharmacia Biotech).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This study was supported by NIH Grant HD31566 (AJWH).
Received for publication May 12, 1997. Revision received August 11, 1997. Accepted for publication August 21, 1997.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Y. Zhu, G.-Y. Yang, B. Ahlemeyer, L. Pang, X.-M. Che, C. Culmsee, S. Klumpp, and J. Krieglstein Transforming Growth Factor-beta 1 Increases Bad Phosphorylation and Protects Neurons Against Damage J. Neurosci., May 15, 2002; 22(10): 3898 - 3909. [Abstract] [Full Text] [PDF]
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