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Western blot analysis of extracts from HeLa cells, transfected with 100 nM SignalSilence® Control siRNA (Fluorescein Conjugate) #6201 (-) or SignalSilence® Bad siRNA I (+), using Bad Antibody and p42 MAPK Antibody #9108. The Bad antibody confirms silencing of Bad expression, while the p42 MAPK antibody is used to control for loading and specificity of Bad siRNA.

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

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Western blot analysis of extracts from 293 cells transfected with Wild-type Bad, Bad (S112A), Bad (S136A) or Bad (S112A/S136A), untreated, TPA-treated or forskolin-treated, using Phospho-Bad (Ser112) Antibody #9291 (top), Phospho-Bad (Ser136) Antibody (middle) or Bad Antibody #9292 (bottom).

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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.

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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.

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Western blot analysis of extracts from HeLa cells, transfected with 100 nM SignalSilence® Control siRNA (Fluorescein Conjugate) #6201 (-) or SignalSilence® Bad siRNA II (+), using Bad Antibody and α-Tubulin (11H10) Rabbit mAb #2125. The Bad antibody confirms silencing of Bad expression, while the α-Tubulin (11H10) Rabbit mAb is used to control for loading and specificity of Bad siRNA.

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Western blot analysis of extracts from COS cells, untreated or TPA-treated, using Bad Antibody #9292 (right) or Phospho-Bad (Ser112) Antibody (left).

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Western blot analysis of GST-Bad, phosphorylated by CKII or PKA in vitro, using Phospho-Bad (Ser112) Antibody #9291 (top), Phospho-Bad (Ser136) Antibody (middle) or Bad Antibody #9292 (bottom).

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

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Western blot analysis of Bad fusion protein phosphorylated by CKII or PKA in vitro, using Phospho-Bad (Ser112) Antibody (upper) or Bad Antibody #9292 (lower).

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Western blot analysis of extracts from 293 cells transfected with Wild-type Bad, Bad (Ser112A), Bad (S136A) or Bad (S112A/S136A), untreated, TPA-treated or forskolin-treated, using Phospho-Bad (Ser112) Antibody #9291 (top), Phospho-Bad (Ser136) Antibody #9295 (middle) or Bad Antibody (bottom).

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Flow cytometric analysis of COS cells, untreated (blue) or Calyculin A/TPA-treated (green), usingPhospho-Bad (Ser112) Antibody compared to Rabbit (DA1E) mAb IgG Isotype Control #3900 (red).

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Image
Product Includes Quantity Applications Reactivity MW(kDa) Isotype
Bad Control Proteins 9293 40 µl
76  
Bad Control Proteins 9293 40 µl
76  
Bad Antibody 9292 100 µl
Western Blotting Immunoprecipitation
H M R Mk 23 Rabbit 
Phospho-Bad (Ser112) Antibody 9291 100 µl
Western Blotting Immunoprecipitation Flow Cytometry
H M R Mk 23 Rabbit 
Phospho-Bad (Ser136) Antibody 9295 200 µl
Western Blotting
M 23 Rabbit 
Anti-rabbit IgG, HRP-linked Antibody 7074 40 µl
Western Blotting
All Goat 
Anti-biotin, HRP-linked Antibody 7075 1 ml
Goat 
20X LumiGLO® Reagent and 20X Peroxide 7003 5 ml each
Western Blotting
 

Specificity / Sensitivity

Phospho-Bad (Ser112) Antibody and Phospho-Bad (Ser136) Antibody detect Bad only when phosphorylated at serine 112 or serine 136, respectively. Bad antibody detects endogenous levels of total Bad protein. These antibodies do not cross-react with related family members. It is necessary to overexpress Bad to detect Ser136 phosphorylation.


Bad is a proapoptotic member of the Bcl-2 family that promotes cell death by displacing Bax from binding to Bcl-2 and Bcl-xL (1,2). Survival factors, such as IL-3, inhibit the apoptotic activity of Bad by activating intracellular signaling pathways that result in the phosphorylation of Bad at Ser112 and Ser136 (2). Phosphorylation at these sites promotes binding of Bad to 14-3-3 proteins to prevent an association between Bad with Bcl-2 and Bcl-xL (2). Akt phosphorylates Bad at Ser136 to promote cell survival (3,4). Bad is phosphorylated at Ser112 both in vivo and in vitro by p90RSK (5,6) and mitochondria-anchored PKA (7). Phosphorylation at Ser155 in the BH3 domain by PKA plays a critical role in blocking the dimerization of Bad and Bcl-xL (8-10).


1.  Yang, E. et al. (1995) Cell 80, 285-291.

2.  Zha, J. et al. (1996) Cell 87, 619-628.

3.  Datta, S.R. et al. (1997) Cell 91, 231-241.

4.  Peso, L. et al. (1997) Science 278, 687-689.

5.  Bonni, A. et al. (1999) Science 286, 1358-1362.

6.  Tan, Y. et al. (1999) J. Biol. Chem. 274, 34859-34867.

7.  Harada, H. et al. (1999) Mol. Cell 3, 413-422.

8.  Tan, Y. et al. (2000) J. Biol. Chem. 275, 25865-25869.

9.  Lizcano, J. et al. (2000) Biochem. J. 349, 547-557.

10.  Datta, S. et al. (2000) Mol. Cell 6, 41-51.


Entrez-Gene Id 12015
Swiss-Prot Acc. Q61337


For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology® is a trademark of Cell Signaling Technology, Inc.
PhosphoPlus® is a trademark of Cell Signaling Technology, Inc.
LumiGLO® is a registered trademark of Kirkegaard & Perry Laboratories.