Immunological demonstration of protein kinase C mu in murine tissues and various cell lines. Differential recognition of phosphorylated forms and lack of down-regulation upon 12-O-tetradecanoylphorphol-13-acetate treatment of cells.
13 murine tissues and 12 cell lines were tested for the expression of the novel protein kinase C (PKC) isoenzyme mu. Using two different PKC mu antibodies (sc-639 and P26720), PKC mu was detected in all tissues and cells and thus proved to be an ubiquitous PKC isotype. However, in some tissues, PKC mu was recognized only by the antibody P26720 and not by sc-639. Thymus, lung and peripheral blood mononuclear cells expressed the greatest amount of PKC mu. Recognition of PKC mu 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 PKC mu. The lack of a decrease in the PKC mu amount and, thus, the lack of down-regulation could be proved using the antibody P26720. This antibody was able to recognize PKC mu 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 PKC mu caused its reduced interaction with the antibody sc-639. Therefore, this antibody might present a well suited tool for the detection of activated PKC mu in 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 PKC mu.
Rennecke
J
J
German Cancer Research Center, Heidelberg, Germany.
Johannes
F J
FJ
Richter
K H
KH
Kittstein
W
W
Marks
F
F
Gschwendt
M
M
D000818:Animals; D000906:Antibodies; D002460:Cell Line; D005260:Female; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D015151:Immunoblotting; D051379:Mice; D008815:Mice, Inbred Strains; D009928:Organ Specificity; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000032:analysis / Q000096:biosynthesis*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D013950:Thymus Gland / Q000201:enzymology
D000906:Antibodies; D010750:Phosphoproteins; C089212:protein kinase D; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate