Cell Signaling Technology

Product Pathways - Ca / cAMP / Lipid Signaling

PKA C-α Antibody #4782

Applications Reactivity Sensitivity MW (kDa) Source
W IP IF-IC F H M R Endogenous 42 Rabbit

Applications Key:  W=Western Blotting  IP=Immunoprecipitation  IF-IC=Immunofluorescence (Immunocytochemistry)  F=Flow Cytometry
Reactivity Key:  H=Human  M=Mouse  R=Rat
Species cross-reactivity is determined by western blot. Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Protocols

Specificity / Sensitivity

PKA C-α Antibody detects endogenous levels of total PKA C-α.

Source / Purification

Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to the carboxy terminal sequence of human PKA C-α. Antibodies are purified by protein A and peptide affinity chromatography.

Western Blotting

Western Blotting

Western blot analysis of extracts from HeLa, C6, PC12 and NIH/3T3 cells, using PKA C-α Antibody.

Western Blotting

Western Blotting

Western blot analysis of extracts from HeLa cells transfected with non-targeted (-) or PKA C-α (+) siRNA. PKA C-α was detected using the PKA C-α Antibody #4782, and Akt1 was detected using Akt1 (2H10) Monoclonal Antibody #2967. The PKA C-α Antibody confirms silencing of PKA C-α expression, and the Akt1 Antibody is used to control for loading and specificity of PKA C-α siRNA.

Western Blotting

Western Blotting

Western blot analysis of extracts from HeLa cells, transfected with either 100 nM SignalSilence® Control siRNA (Fluorescein Conjugate) #6201 (-), SignalSilence® PKA C-α siRNA II (+) or SignalSilence® PKA C-α siRNA I #6406 (+), using PKA C-α Antibody #4782 and α-Tubulin (11H10) Rabbit mAb #2125. The PKA C-α antibody confirms silencing of PKA C-α expression and α-Tubulin (11H10) Rabbit mAb is used to control for loading and specificity of PKA C-α siRNA.


Flow Cytometry

Flow Cytometry

Flow cytometric analysis of HeLa cells, using PKA C-α Antibody (blue) compared to a nonspecific negative control antibody (red).

IF-IC

IF-IC

Confocal immunofluorescent analysis of HeLa cells using PKA C-alpha Antibody (green). Actin filaments have been labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5™ (fluorescent DNA dye).

Background

The second messenger cyclic AMP (cAMP) activates cAMP-dependent protein kinase (PKA or cAPK) in mammalian cells and controls many cellular mechanisms such as gene transcription, ion transport, and protein phosphorylation (1). Inactive PKA is a heterotetramer composed of a regulatory subunit (R) dimer and a catalytic subunit (C) dimer. In this inactive state, the pseudosubstrate sequences on the R subunits block the active sites on the C subunits. Three C subunit isoforms (C-α, C-β, and C-γ) and two families of regulatory subunits (RI and RII) with distinct cAMP binding properties have been identified. The two R families exist in two isoforms, α and β (RI-α, RI-β, RII-α, and RII-β). Upon binding of cAMP to the R subunits, the autoinhibitory contact is eased and active monomeric C subunits are released. PKA shares substrate specificity with Akt (PKB) and PKC, which are characterized by an arginine at position -3 relative to the phosphorylated serine or threonine residue (2). Substrates that present this consensus sequence and have been shown to be phosphorylated by PKA are Bad (Ser155), CREB (Ser133), and GSK-3 (GSK-3α Ser21 and GSK-3β Ser9) (3-5). In addition, combined knock-down of PKA C-α and -β blocks cAMP-mediated phosphorylation of Raf (Ser43 and Ser259) (6). Autophosphorylation and phosphorylation by PDK-1 are two known mechanisms responsible for phosphorylation of the C subunit at Thr197 (7).

  1. Montminy, M. (1997) Annu. Rev. Biochem. 66, 807-822.
  2. Dell'Acqua, M.L. and Scott, J.D. (1997) J. Biol. Chem. 272, 12881-12884.
  3. Tan, Y. et al. (2000) J. Biol. Chem. 275, 25865-25869.
  4. Gonzalez, G.A. and Montminy, M.R. (1989) Cell 59, 675-680.
  5. Fang, X. et al. (2000) Proc. Natl. Acad. Sci. USA 97, 11960-11965.
  6. Dumaz, N. and Marais, R. (2003) J. Biol. Chem. 278, 29819 -29823.
  7. Moore, M.J. et al. (2002) J. Biol. Chem. 277, 47878-47884.

Application References

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This product is intended for research purposes only. The product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

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