Immunological Demonstration of Protein Kinase Cμ in Murine Tissues and Various Cell Lines

Open Access
Publisher Website
Google Scholar
Abstract
13 murine tissues and 12 cell lines were tested for the expression of the novel protein kinase C (PKC) isoenzyme μ. Using two different PKCμ antibodies (sc-639 and P26720), PKCμ was detected in all tissues and cells and thus proved to be an ubiquitous PKC isotype. However, in some tissues, PKCμ was recognized only by the antibody P26720 and not by sc-639. Thymus, lung and peripheral blood mononuclear cells expressed the greatest amount of PKCμ. Recognition of PKCμ by the antibody sc-639 was drastically impaired when treating keratinocytes or mouse skin in vivo with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), thus mimicking down-regulation of PKCμ. The lack of a decrease in the PKCμ amount and, thus, the lack of down-regulation could be proved using the antibody P26720. This antibody was able to recognize PKCμ in extracts of untreated as well as TPA-treated tissues or cells. Phosphorylation of proteins in a cell-free system (cell or tissue extracts) in the presence and absence of TPA or other PKC activators and various protein kinase inhibitors indicated that phosphorylation of activated PKCμ caused its reduced interaction with the antibody sc-639. Therefore, this antibody might present a well suited tool for the detection of activated PKCμin vivo. Moreover, our results clearly show that some antibodies, such as sc-639, might be able to selectively detect non-phosphorylated or phosphorylated forms of a protein, and that such properties of an antibody have to be studied carefully before the latter can be used for reliable quantitative determination of this protein. We consider this information important to avoid misinterpretation of data concerning the immunological quantification of proteins such as PKCμ.