Product Pathways - Ca / cAMP / Lipid Signaling

Phospho-PKC Antibody Sampler Kit #9921

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Applications Key:  W=Western Blotting  IP=Immunoprecipitation  IHC-P=Immunohistochemistry (Paraffin)  F=Flow Cytometry
Reactivity Key:  H=Human  M=Mouse  R=Rat  Mk=Monkey  Hm=Hamster  B=Bovine

Specificity / Sensitivity

Phospho-PKC (pan) (betaII Ser660) Antibody detects PKCalpha, beta I, beta II, delta, epsilon and eta isoforms only when phosphorylated at a carboxy-terminal residue homologous to serine 660 of PKCbeta II. Phospho-PKCdelta (Ser643) Antibody detects PKC delta when phosphorylated at serine 643, and PKCtheta when phosphorylated at serine 676. PKD/PKCmu antibody detects PKD/PKCmu only. All other phospho-PKC antibodies recognize only their specific isoform when phosphorylated at the indicated sites.

Western Blotting

Western blot analysis of extracts from PDGF (50 ng/ml) or TPA (0.2 µM) treated NIH/3T3 cells, using Phospho-PKD/PKCµ (Ser916) Antibody #2051 (upper) and PKD/PKCµ Antibody #2052 (lower).

Western Blotting

Western blot analysis of extracts from PDGF (50 ng/ml) or TPA (0.2 µM) treated NIH/3T3 cells, using Phospho-PKD/PKCµ (Ser744/748) Antibody #2054 (upper) and PKD/PKCµ Antibody #2052 (lower).

Western Blotting

Western blot analysis of Baculovirus expressed PKCbeta and PKCbeta Ser660/Ala mutant, using Phospho-PKC (pan) (betaII Ser660) Antibody #9371 (upper) or PKCbeta antibody (lower).

Western Blotting

Western blot analysis of extracts from TPA, G?6983 or Bisindolylmaleimide treated 293 cells, using Phospho-PKC (pan) (betaII Ser660) Antibody #9371.

Western Blotting

Western blot analysis of extracts from Baculovirus expressed PKC isoforms alpha, beta, gamma, delta, epsilon, using Phospho-PKCdelta (Thr505) Antibody #9374 (upper) or control PKCalpha, beta, gamma, delta, epsilon antibodies (lower).

Western Blotting

Western blot analysis of extracts from TPA treated (0.2 µM) U-937 cells, using Phospho-PKCdelta (Thr505) Antibody #9374.

Western Blotting

Western blot analysis of extracts from NIH/3T3 cells, untreated or TPA-treated (0.2 µM), using Phospho-PKCalpha/betaII (Thr638/641) Antibody #9375.

Western Blotting

Western blot analysis of Baculovirus expressed PKC isoforms alpha, beta, gamma, delta, epsilon treated with or without lambda protein phosphatase, using Phospho-PKCalpha/betaII (Thr638/641) Antibody #9375 (upper) or PKCalpha, beta, gamma, delta, epsilon antibodies (lower).

Western Blotting

Western blot analysis of Baculovirus expressed PKC isoforms alpha, beta, gamma, delta, epsilon treated with or without lambda protein phosphatase, using Phospho-PKCdelta (Ser643) Antibody #9376 (upper) and PKCalpha, beta, gamma, delta, epsilon antibodies (lower).

Western Blotting

Western blot analysis of U-937 cell extracts treated with 0.2 µM TPA, using Phospho-PKCdelta (Ser643) Antibody #9376.

Western Blotting

Western blot analysis of extracts from TPA (0.2 µM) treated Jurkat cells for 0 and 30 minutes (lanes 1 and 2) and immunoprecipitates from Jurkat cells by PKCtheta antibody treated with or without l phosphatase (lanes 3 and 4), using Phospho-PKCtheta (Thr538) Antibody #9377 (upper) and PKCtheta antibody (lower).

Western Blotting

Western blot analysis of extracts from EGF treated (100 ng/ml) 293 cells or lambda phosphatase (lambda-PPase) treated purified PKCzeta, using Phospho-PKCzeta/lambda (Thr410/403) Antibody #9378 (upper) or PKCzeta antibody (lower).

Source / Purification

Polyclonal antibodies are produced by immunizing rabbits with synthetic phospho-peptides (KLH-coupled) derived from the sequence of the human protein PKCbeta II, PKCalpha, PKCdelta, PKCtheta, PKCzeta or the mouse protein (PKD). Antibodies are purified by protein A and peptide affinity chromatography.

Background

Activation of protein kinase C (PKC) is one of the earliest events in a cascade that controls a variety of cellular responses, including secretion, gene expression, proliferation and muscle contraction (1,2). PKC isoforms belong to three groups based on calcium dependency and activators. Classical PKCs are calcium-dependent via their C2 domains and are activated by phosphatidylserine (PS), diacylglycerol (DAG) and phorbol esters (TPA, PMA) through their cysteine-rich C1 domains. Both novel and atypical PKCs are calcium-independent, but only novel PKCs are activated by PS, DAG and phorbol esters (3-5). Members of these three PKC groups contain a pseudo-substrate or autoinhibitory domain that binds to substrate-binding site in the catalytic domain to prevent activation in the absence of cofactors or activators.Control of PKC activity is regulated through three distinct phosphorylation events. Phosphorylation of Thr500 in the activation loop, the autophosphorylation site at Thr641 and at carboxy-terminal hydrophobic site Ser660 occurs in vivo (2). Atypical PKC isoforms lack hydrophobic region phosphorylation, which correlates with the presence of glutamic acid rather than the serine or threonine residues found in more typical PKC isoforms. Either the enzyme PDK1 or a close relative is responsible for PKC activation.A recent addition to the PKC superfamily is PKCμ (PKD), which is regulated by DAG and TPA through its C1 domain. PKD is distinguished by the presence of a PH domain and by its unique substrate recognition and Golgi localization (6). PKC-related kinases (PRK) lack the C1 domain and do not respond to DAG or phorbol esters. Phosphatidylinositol lipids activate PRKs and small Rho-family GTPases bind to the homology region 1 (HR1) to regulate PRK kinase activity (7).

1. Nishizuka, Y. (1984) Nature 308, 693-698.
2. Keranen, L.M. et al. (1995) Curr. Biol. 5, 1394-1403.
3. Mellor, H. and Parker, P.J. (1998) Biochem J. 332 (Pt 2), 281-292.
4. Ron, D. and Kazanietz, M.G. (1999) FASEB J. 13, 1658-1676.
5. Moscat, J. and Diaz-Meco, M.T. (2000) EMBO Rep. 1, 399-403.
6. Baron, C.L. and Malhotra, V. (2002) Science 295, 325-328.
7. Flynn, P. et al. (2000) J. Biol. Chem. 275, 11064-11070.

Application References

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