This antibody reacts with PKC lambda immunoprecipitates, indicating cross-reactivity for PKC lambda [pT563]. PKC zeta [pT560] (83% homologous) has been shown to cross-react by peptide competition. Peptide competition also suggests that this antibody may partially cross-react with PKC beta 1 [pS642] (58% homologous) and PKC nu [pT655] (42% homologous).
The antibody has been negatively preadsorbed using a non-phosphopeptide corresponding to the site of phosphorylation to remove antibody that is reactive with non-phosphorylated PKC iota. The final product is generated by affinity chromatography using a PKC iota-derived peptide that is phosphorylated at threonine 555.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use 3-5µg for 10 cells.
1/1000. Detects a band of approximately 76 kDa.
Calcium-independent, phospholipid-dependent, serine- and threonine-specific kinase. May play a role in the secretory response to nutrients. Involved in cell polarization processes and the formation of epithelial tight junctions. Implicated in the activation of several signaling pathways including Ras, c-Src and NF-kappa-B pathways. Functions in both pro- and anti-apoptotic pathways. Functions in the RAC1/ERK signaling required for transformed growth. Plays a role in microtubule dynamics through interaction with RAB2A and GAPDH and recruitment to vesicular tubular clusters (VTCs).
Predominantly expressed in lung and brain, but also expressed at lower levels in many tissues including pancreatic islets. Highly expressed in non-small cell lung cancers.
Belongs to the protein kinase superfamily. AGC Ser/Thr protein kinase family. PKC subfamily. Contains 1 AGC-kinase C-terminal domain. Contains 1 OPR domain. Contains 1 phorbol-ester/DAG-type zinc finger. Contains 1 protein kinase domain.
The OPR domain mediates interaction with SQSTM1. The C1 domain does not bind diacylglycerol (DAG).
On neuronal growth factor (NGF) stimulation, phosphorylated by Src on Tyr-265, Tyr-280 and Tyr-334. Phosphorylation on Tyr-265 facilitates binding to KPNB1/importin-beta regulating entry of PRKCI into the nucleus. Phosphorylation on Tyr-334 is important for NF-kappa-B stimulation.
Cytoplasm. Membrane. Endosome. Nucleus. Transported into the endosome through interaction with SQSTM1/p62. After phosphorylation by cSrc, transported into the nucleus through interaction with KPNB1. Colocalizes with CDK7 in the cytoplasm and nucleus. Vesicular tubular clusters. Transported to VTCs through interaction with RAB2A.
Western blot - Anti-PKC iota (phospho T555 + T563) antibody (ab5813)
Peptide Competition and Phosphatase Treatment: Lysates prepared from Jurkat cells stimulated with PMA were resolved by SDS-PAGE on a 10% polyacrylamide gel and transferred to PVDF. Membranes were either left untreated (1-4) or treated with lambda phosphatase (5), blocked with a 5% low-fat milk-TBST buffer for one hour at room temperature, and incubated with ab5813 antibody for two hours at room temperature in a 3% low-fat milk-TBST buffer, following prior incubation with: no peptide (1), the non-phosphopeptide corresponding to the immunogen (2), a generic phosphothreonine-containing peptide (3), or, the phosphopeptide immunogen (4). After washing, membranes were incubated with goat F(ab’)2 anti-rabbit IgG HRP conjugate and bands were detected using the Pierce SuperSignalTM method. The data show that the phosphopeptide corresponding to PKC iota [pT555] blocks the antibody signal. The peptide corresponding to PKC zeta [pT560] blocks the antibody signal and the peptides corresponding to PKC isoforms beta 1 [pT642] and gamma [pÔ655] partially block the antibody signal (data not shown), suggesting cross-reactivity of the antibody with these sites. The antibody signal was not blocked by the corresponding peptides of any other PKC isoforms. The data also show that phosphatase stripping eliminates the signal, verifying that the antibody is phospho-specific.
Vichas A et al. The Ski2-family helicase Obelus regulates Crumbs alternative splicing and cell polarity. J Cell Biol211:1011-24 (2015).
Read more (PubMed: 26644515) »
Qian J et al. The RNA binding protein FXR1 is a new driver in the 3q26-29 amplicon and predicts poor prognosis in human cancers. Proc Natl Acad Sci U S A112:3469-74 (2015).
Read more (PubMed: 25733852) »