Quantitation of protein kinase C by immunoblot--expression in different cell lines and response to phorbol esters.
Antisera have been raised against human protein kinase C and also against a synthetic peptide based on the sequence of the bovine brain enzyme (LLNQEE-GEYYNVPIPE). These antibodies react with protein kinase C from a number of species (human, murine, rat, rabbit, bovine), indicating substantial conservation of epitopes. These antisera have been used to quantitate directly protein kinase C by immunoblot analysis. We show here that there is a strict correlation between the levels of immunoreactive polypeptide and extractable calcium- and phospholipid-dependent kinase activity for various cell lines. Treatment of murine, rat, and human cells with phorbol dibutyrate was found to deplete levels of immunoreactive protein kinase C severely. A detailed study of the time course of this depletion in Swiss 3T3 cells shows that it follows precisely the loss of extractable activity. On exposure to 400 nM phorbol 12,13-dibutyrate protein kinase C was essentially undetectable by 40 hours; the half-life of this down-regulation was 6.7 hours. This data thus demonstrate that the loss of immunoreactive protein kinase C and of extractable calcium- and phospholipid-dependent kinase activity precisely parallels the phorbol ester induced down-regulation of binding and responsiveness in Swiss 3T3 cells.
D000818:Animals; D000936:Antigen-Antibody Complex; D002417:Cattle; D002460:Cell Line; D005910:Glioma; D006801:Humans; D007158:Immunologic Techniques; D051379:Mice; D009447:Neuroblastoma; D015240:Phorbol 12,13-Dibutyrate; D010703:Phorbol Esters / Q000494:pharmacology*; D011493:Protein Kinase C / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism*; D011817:Rabbits; D051381:Rats; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D000936:Antigen-Antibody Complex; D010703:Phorbol Esters; D015240:Phorbol 12,13-Dibutyrate; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate