Western blot analysis of extracts from NIH/3T3 cells, untreated, PDGF-treated (50 ng/ml) or TPA-treated (0.2 µM), using Phospho-PKD/PKCµ (Ser916) Antibody (upper) or PKD/PKCµ Antibody #2052 (lower).
Western blot analysis of extracts from various cell lines using PKD/PKCμ (D4J1N) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
Western blot analysis of extracts from NIH/3T3 cells, untreated, PDGF-treated (50 ng/ml) or TPA-treated (0.2 µM), using Phospho-PKD/PKCµ (Ser744/748) Antibody (upper) or PKD/PKCµ Antibody #2052 (lower).
Western blot analysis of Baculovirus expressed PKCβ and PKCβ Ser660/Ala mutant, using Phospho-PKC (pan) (βII Ser660) Antibody (upper) or control PKCβ antibody (lower).
Western blot analysis of extracts from U-937 cells, untreated or TPA treated (0.2 µM), using Phospho-PKCδ (Thr505) Antibody.
Immunohistochemical analysis of paraffin-embedded human breast carcinoma, using Phospho-PKCdelta/theta (Ser643/676) Antibody.
Western blot analysis of extracts from 293 cells, untreated or EGF-treated (100 ng/ml) and purified PKCzeta, untreated or lambda phosphatase-treated, using Phospho-PKCzeta/lambda (Thr410/403) Antibody (upper) or PKCzeta antibody (lower).
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 TPA, Go6983 and/or Bisindolylmaleimide treated 293 cells, using Phospho-PKC (pan) (βII Ser660) Antibody.
Western blot analysis of Baculovirus-expressed PKC isoforms alpha, beta, gamma, delta and epsilon, untreated or treated with lambda protein phosphatase, using Phospho-PKCalpha/beta II (Thr638/641) Antibody (upper) or PKCalpha, beta, gamma, delta, epsilon antibodies (lower).
Western blot analysis of extracts from Baculovirus-infected cells expressing PKC isoforms α, β, γ, δ or ε, using Phospho-PKCδ (Thr505) Antibody (upper) or control PKCα, β, γ, δ, ε antibodies (lower).
Immunohistochemical analysis of paraffin-embedded human lung carcinoma, using Phospho-PKCdelta/theta (Ser643/676). Antibody.
Western blot analysis of extracts from 293 cells, untreated (lane 1), TPA-treated (.2μM for 20 minutes) (lanes 2 & 3), and CIP/λ phosphatase treated (lane 3), using Phospho-PKCθ (Thr538) Antibody #9377, PKCθ Antibody #2059, and β-actin Antibody #4967.
Western blot analysis of Baculovirus expressed PKC isoforms, using Phospho-PKC (pan) (βII Ser660) Antibody.
Western blot analysis of extracts from 293 cells, untreated, TPA treated (200 nM), or treated with TPA and CIP and λ phosphatases using Phospho-PKCα/βII (Thr638/641) Antibody or PKCα Antibody #2056.
Western blot analysis of extracts from U-937 cells, untreated or TPA-treated (0.2 µM), using Phospho-PKCdelta/theta (Ser643/676) Antibody.
Western blot analysis of Baculovirus expressed PKC isoforms alpha, beta, gamma, delta and epsilon, untreated or lambda protein phosphatase-treated, using Phospho-PKCdelta/theta (Ser643/676) Antibody (upper) or PKCalpha, -beta, -gamma, -delta and -epsilon antibodies (lower).
|Phospho-PKD/PKCμ (Ser916) Antibody 2051||20 µl||
||H M R Mk||115||Rabbit|
|PKD/PKCμ (D4J1N) Rabbit mAb 90039||20 µl||
||H Mk||115||Rabbit IgG|
|Phospho-PKD/PKCμ (Ser744/748) Antibody 2054||20 µl||
||H M R Mk||115||Rabbit|
|Phospho-PKC (pan) (βII Ser660) Antibody 9371||20 µl||
||H M R Mk||78, 80, 82, 85||Rabbit|
|Phospho-PKCα/β II (Thr638/641) Antibody 9375||20 µl||
||H M Mk||80, 82||Rabbit|
|Phospho-PKCδ (Thr505) Antibody 9374||20 µl||
|Phospho-PKCδ/θ (Ser643/676) Antibody 9376||20 µl||
||H M R Mk X||78||Rabbit|
|Phospho-PKCθ (Thr538) Antibody 9377||20 µl||
||H R Mk||79||Rabbit|
|Phospho-PKCζ/λ (Thr410/403) Antibody 9378||20 µl||
||H M R Mk||76||Rabbit|
|Anti-rabbit IgG, HRP-linked Antibody 7074||100 µl||
The Phospho-PKC Antibody Sampler Kit provides a fast and economical means of evaluating multiple PKC isoforms and their phosphorylation state. The kit contains enough primary and secondary antibodies to perform two Western blot experiments.
Phospho-PKC (pan) (βII Ser660) Antibody detects PKCα, β I, β II, δ, ε and η isoforms only when phosphorylated at a carboxy-terminal residue homologous to Ser660 of PKCβ II. Phospho-PKCδ (Ser643) Antibody detects PKC δ when phosphorylated at Ser643, and PKCθ when phosphorylated at Ser676. PKD/PKCμ (D4J1N) Rabbit mAb detects endogenous levels of PKD/PKCμ only. All other phospho-PKC antibodies recognize only their specific isoform when phosphorylated at the indicated sites.
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of human PKD. Polyclonal antibodies are produced by immunizing animals with synthetic phosphopeptides corresponding to the sequence of the human protein PKCβ II, PKCα, PKCδ, PKCθ, or PKCζ . Antibodies are purified by protein A and peptide affinity chromatography.
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 sites 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 occurs in vivo at Thr500 in the activation loop, at Thr641 through autophosphorylation, and at the carboxy-terminal hydrophobic site Ser660 (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. 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).
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