Product Pathways - Apoptosis / Autophagy

Pro-Apoptosis Bcl-2 Family Antibody Sampler Kit #9942

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Applications Key:  W=Western Blotting  IP=Immunoprecipitation  IHC-P=Immunohistochemistry (Paraffin)  IF-IC=Immunofluorescence (Immunocytochemistry)  F=Flow Cytometry
Reactivity Key:  H=Human  M=Mouse  R=Rat  Mk=Monkey

Specificity / Sensitivity

Each antibody in the Pro-Apoptosis Bcl-2 Family Antibody Sampler Kit recognizes only its specific target. The antibodies do not cross-react with other Bcl-2 family members.

Western Blotting

Western blot analysis of extracts from HT-1080 and NBT-II cells, using Bok Antibody.

Western Blotting

Western blot analysis of extracts from HeLa, K562, HT-29 and HepG2 cells, using Bik Antibody.

Western Blotting

Western blot analysis of extracts from Jurkat, C6 and NIH/3T3 cells, using Bmf Antibody.

Western Blotting

Western blot analysis of extracts from K562, HT1080, PANC1, A204, and SR cells, using Puma Antibody.

Western Blotting

Western blot analysis of extracts from COS cells, untreated or TPA-treated, using Bad Antibody (right) or Phospho-Bad (Ser112) Antibody #9291 (left).

Western Blotting

Western blot analysis of extracts from 293 cells transfected with Wild-type Bad, Bad (Ser112A), Bad (S136A) or Bad (S112A/S136A), untreated or treated with TPA or forskolin as indicated, using Phospho-Bad (Ser112) (7E11) Mouse mAb (upper) or Bad Antibody #9292 (lower).

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded human renal cell carcinoma, showing cytoplasmic localization, using Bok Antibody.

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded human colon carcinoma, using Bik Antibody.

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded human breast carcinoma, showing cytoplasmic localization, using Phospho-Bad (Ser112) (7E11) Mouse mAb.

IF-IC

Immunofluorescent analysis of HeLa cells, showing perinuclear localization, using Puma Antibody.

IF-IC

Confocal immunofluorescent analysis of C2C12 cells labeled with Puma Antibody (left, blue) showing colocalization with mitochondria that have been labeled with MitoTracker® Red CMXRos (red, right). Actin filaments are shown labeled with fluorescein phalloidin (green).

Source / Purification

Monoclonal antibody is produced by immunizing mice with a synthetic phospho-peptide corresponding to residues surrounding Ser112 of mouse Bad. Polyclonal antibodies are produced by immunizing rabbits with synthetic peptides (KLH-coupled) corresponding to residues surrounding Ser112 of mouse Bad, the amino-termini of human Bak, Bik, Bim and Bmf, residues surrounding the central region of human Bok or the carboxy terminus of human Puma. Polyclonal antibodies are purified by protein A and peptide affinity chromatography.

Background

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), which mediate protein interactions. The family can be separated into three groups based upon function and sequence homology: pro-surivival members including Bcl-2, Bcl-xL, Mcl-1, A1 and Bcl-w; pro-apoptotic proteins including 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.

Bad is a pro-apoptotic member of the Bcl-2 family that can displace Bax from binding to Bcl-2 and Bcl-xL, resulting in cell death (6,7). Survival factors such as IL-3 can inhibit the apoptotic activity of Bad by activating intracellular signaling pathways that result in the phosphorylation of Bad at Ser112 and Ser136 (7). Phosphorylation at these sites results in the binding of Bad to 14-3-3 proteins and the inhibition of Bad binding to Bcl-2 and Bcl-xL (7). Akt has been shown to promote cell survival via its ability to phosphorylate Bad at Ser136 (8,9). Ser112 has been shown to be the substrate in vivo and in vitro of p90RSK (10,11) and mitochondria-anchored PKA (12).

1. Cory, S. et al. (2003) Oncogene 22, 8590-8607.
2. Antonsson, B. and Martinou, J. (2000) Exp. Cell Res. 256, 50-57.
3. Sharpe, J.C. et al. (2004) Biochim. Biophys. Acta. 1644, 107-113.
4. Korsmeyer, S.J. et al. (1993) Semin. Cancer Biol. 4, 327-337.
5. Bouillet, P. and Strasser, A. (2002) J. Cell Sci. 115, 1567-1574.
6. Yang, E. et al. (1995) Cell 80, 285-291.
7. Zha, J. et al. (1996) Cell 87, 619-628.
8. Datta, S. R. et al. (1997) Cell 91, 231-241.
9. Peso, L. et al. (1997) Science 278, 687-689.
10. Bonni, A. et al. (1999) Science 286, 1358-1362.
11. Tan, Y. et al. (1999) J. Bio. Chem. 274, 34859-34867.
12. Harada, H. et al. (1999) Mol. Cell 3, 413-422.

Application References

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