Flow cytometric analysis of Jurkat cells using Phospho-Bcl-2 (Ser70) (5H2) Rabbit mAb and Propidium Iodide (PI)/RNase Staining Solution #4087 to measure DNA content. Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
Western blot analysis of Jurkat cells, untreated or treated with paclitaxel for the indicated times, using Phospho-Bcl-2 (Thr56) Antibody (Human Specific) (top) or Bcl-2 Antibody (Human Specific) #2872 (bottom).
Western blot analysis of control HeLa cells (lane 1) or Bcl-2 knockout HeLa cells (lane 2) using Bcl-2 (D55G8) Rabbit mAb #4223 (upper), or β-actin (13E5) Rabbit mAb #4970 (lower). The absence of signal in the Bcl-2 knockout HeLa cells confirms the specificity of the antibody for Bcl-2.
Flow cytometric analysis of untreated Jurkat cells, using Bcl-xL (54H6) Rabbit mAb (blue) compared to a nonspecific negative control antibody (red).
Western blot analysis of extracts from various cell lines using Mcl-1 (D35A5) Rabbit mAb.
After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO® is added and emits light during enzyme catalyzed decomposition.
Western blot analysis of extracts from Jurkat cells, untreated or treated with paclitaxel (1 μM, overnight) and with or without λ phosphatase, using Phospho-Bcl-2 (Ser70) (5H2) Rabbit mAb (upper) or Bcl-2 #2876 (lower).
Western blot analysis of extracts from various cell lines using Bcl-2 (D55G8) Rabbit mAb (Human Specific).
Confocal immunofluorescent analysis of HeLa cells using Bcl-xL (54H6) Rabbit mAb (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
Western blot analysis of extracts from 293T cells, mock transfected or transfected with human or mouse Mcl-1 constructs, using Mcl-1 (D35A5) Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded human lung carcinoma, using Bcl-xL (54H6) Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded human colon carcinoma, using Bcl-xL (54H6) Rabbit mAb in the presence of control peptide (left) or Bcl-xL Blocking Peptide #1225 (right).
Immunohistochemical analysis of paraffin-embedded human prostate carcinoma, showing cytoplasmic localization, using Bcl-xL (54H6) Rabbit mAb.
Immunoprecipitation of Bcl-xL from Jurkat cell extracts, using Bcl-xL (54H6) Rabbit mAb. Lane 1 is the lysate control, lane 2 is antibody alone and lane 3 is antibody plus lysate.
Western blot analysis of extracts from Jurkat and HeLa (human), COS (monkey), NIH/3T3 and L929 (mouse), and PC12 and C6 (rat) cells, using Bcl-xL (54H6) Rabbit mAb.
Western blot analysis of extracts from HeLa cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-), SignalSilence® BcL-xL siRNA I (+) or SignalSilence® Bcl-xL siRNA II #6363 (+), using Bcl-xL (54H6) Rabbit mAb #2764 (upper) or α-Tubulin (11H10) Rabbit mAb #2125 (lower). The Bcl-xL (54H6) Rabbit mAb confirms silencing of Bcl-xL expression, while the α-Tubulin (11H10) Rabbit mAb is used as a loading control.
|Phospho-Bcl-2 (Ser70) (5H2) Rabbit mAb 2827||20 µl||
|Phospho-Bcl-2 (Thr56) Antibody (Human Specific) 2875||20 µl||
|Bcl-2 (D55G8) Rabbit mAb (Human Specific) 4223||20 µl||
|Bcl-xL (54H6) Rabbit mAb 2764||20 µl||
||H M R Mk||30||Rabbit IgG|
|Mcl-1 (D35A5) Rabbit mAb 5453||20 µl||
||H M Mk||40 (human), 35 (mouse)||Rabbit IgG|
|Anti-rabbit IgG, HRP-linked Antibody 7074||100 µl||
The Pro-Survival Bcl-2 Family Antibody Sampler Kit provides an economical means to examine several members of the Bcl-2 family. The kit contains enough primary and secondary antibodies to perform two western blot experiments.
Each antibody in the Pro-Survival Bcl-2 Family Antibody Sampler Kit recognizes only its specific target. The antibodies do not cross-react with other Bcl-2 family members. Phospho-Bcl-2 (Ser70) (5H2) Rabbit mAb detects endogenous levels of human Bcl-2 only when phosphorylated at Ser70. Phospho-Bcl-2 (Thr56) Antibody (Human Specific) detects endogenous levels of human Bcl-2 only when phosphorylated at Thr56.
Total Mcl-1, Bcl-xL, and Bcl-2 monoclonal antibodies are produced by immunizing animals with synthetic peptides corresponding to residues surrounding Leu210 of human Mcl-1, Asp61 of human Bcl-xL, and Gly47 of human Bcl-2. Phospho-Bcl-2 (Ser70) (5H2) Rabbit mAb is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser70 of human Bcl-2. Phospho-Bcl-2 (Thr56) Antibody (Human Specific) is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Thr56 of human Bcl-2. Polyclonal antibodies are purified by protein A and peptide affinity chromatography.
The Bcl-2 family consists of a number of evolutionarily conserved proteins containing Bcl-2 homology domains (BH) that regulate apoptosis through control of mitochondrial membrane permeability and release of cytochrome c (1-3). Four BH domains have been identified (BH1-4) that mediate protein interactions. The family can be separated into three groups based upon function and sequence homology: pro-survival members include Bcl-2, Bcl-xL, Mcl-1, A1 and Bcl-w; pro-apoptotic proteins include Bax, Bak and Bok; and "BH3 only" proteins Bad, Bik, Bid, Puma, Bim, Bmf, Noxa and Hrk. Interactions between death-promoting and death-suppressing Bcl-2 family members has led to a rheostat model in which the ratio of pro-apoptotic and anti-apoptotic proteins controls cell fate (4). Thus, pro-survival members exert their behavior by binding to and antagonizing death-promoting members. In general, the "BH3-only members" can bind to and antagonize the pro-survival proteins leading to increased apoptosis (5). While some redundancy of this system likely exists, tissue specificity, transcriptional and post-translational regulation of many of these family members can account for distinct physiological roles.
Several phosphorylation sites have been identified within Bcl-2 including Thr56, Ser70, Thr74 and Ser87 (6). These phosphorylation sites may be targets of the ASK1/MKK7/JNK1 pathway, and phosphorylation of Bcl-2 may be a marker for mitotic events (7,8). Mutation of Bcl-2 at Thr56 or Ser87 inhibits its anti-apoptotic activity during glucocorticoid-induced apoptosis of T lymphocytes (9). Interleukin 3 and JNK-induced Bcl-2 phosphorylation at Ser70 may be required for its enhanced antiapoptotic functions (10).
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