Immunoblotting PKC-delta: a cautionary note from the bench

Antibodies that specifically recognize signaling proteins (or individual phosphorylation events at specific residues in proteins of interest) have become important tools in the study of signaling pathways. However, the recognition properties of many commercially available antibodies have not been fully characterized. In the course of studies exploring PKC- phosphorylation mechanisms in cardiomyocytes, we have demonstrated that a BD Transduction Laboratories anti-PKC- MAb (generally viewed as an anti-PKC- protein antibody) recognizes PKC- in resting, but not in PMA-treated, cardiomyocytes. The observations that PKC- immunoreactivity is preserved when cultures are treated with PMA in the presence of a the PKC inhibitor GF-109203X and that PKC- immunoreactivity is restored by in vitro acid phosphatase treatment indicate that the epitope recognized by the BD Transduction Laboratories anti-PKC- MAb is masked by phosphorylation. The BD Transduction Laboratories MAb is poorly suited for studies that compare PKC- expression in resting and agonist-activated samples (or in studies of the relationship between PKC- phosphorylation and PKC- downregulation) because it artifactually displays PKC- phosphorylation as a decline in total PKC- protein. Other studies have shown that two anti-PKC--pY³¹¹ antibodies (manufactured by Cell Signaling Technology, Beverly, MA, and BioSource International, Camarillo, CA, respectively) specifically recognize stimulus-induced changes in PKC--Y³¹¹ phosphorylation on the endogenous PKC- enzyme, but the Cell Signaling Technology anti-PKC--pY³¹¹ antibody provides a better measure of Y³¹¹ phosphorylation in overexpressed PKC-. Collectively, these studies have identified features of anti-PKC- antibodies that affect the interpretation of immunoblot analysis experiments. These findings related to PKC- may be symptomatic of a more pervasive feature of immunoblot analysis studies of phosphoproteins in general. protein phosphorylation; signal transduction pathways; cardiomyocytes