Immunoblotting PKC-δ: a cautionary note from the bench

Abstract

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

Regulation of protein kinase Cδ downregulation by protein kinase Cε and mammalian target of rapamycin complex 2

“…In fact, PKCδ level detected using either monoclonal or polyclonal antibody to PKCδ was equivalent in cells treated with Gö 6983. These results suggest that the immunoreactivity of monoclonal antibody to PKCδ is decreased when PKCδ is phosphorylated at T505 site as has been reported [30]. …”

“…On the other hand, phosphorylation of PKCδ at Y311 was reported to be important for downregulation of PKCδ by Src but not by phorbol esters [29]. It is now realized that the immunoreactivity of the PKCδ antibody used in these studies is altered by the PDBu treatment [30]. In addition, some of the studies on PKCδ phosphorylation and downregulation were performed in serum-deprived adherent cells grown in suspension [23] and the regulation of PKCs in suspension culture could be distinct from adherent cells [31].…”

Structural Basis of Protein Kinase C Isoform Function

“…However, we recently reported that a commercially available anti-PKC δ monoclonal antibody (from BD Transduction Laboratories, that has been used widely for almost a decade to detect and immunoprecipitate PKC δ ) recognizes PKC δ in resting cardiomyocytes, but artifactually perceives a decline in PKC immunoreactivity under conditions of PMA activation (without any change in PKC δ protein expression, Ref. 174). We showed that PKC δ immunoreactivity is preserved when in vivo PMA treatment is performed in the presence of the PKC inhibitor GF109203X and that PKC δ immunoreactivity is restored by in vitro acid phosphatase treatment, suggesting the recognition epitope for the BD Transduction Laboratory anti-PKC δ antibody encompasses a site for PKC-dependent phosphorylation (or a site that is concealed as a result of a PKC-dependent phosphorylation elsewhere in the protein that alters conformation).…”

“…While antibodies are generally assumed to recognize all of the enzyme in the cell (regardless of posttranslational modifications), recent experience in our laboratory and by others suggests otherwise. Many anti-PKC antibodies (particularly for PKC δ ) do not quantitatively recover all of the immunoreactive enzyme from a cell lysate (at least in part because antibodies that interact with PKCs in a conformationally sensitive manner may be considerably more common than heretofore appreciated) (174). Antibody bias that can lead to the selective recovery of pools of enzyme with distinct posttranslational modifications and catalytic properties; this would seriously undermine the results of an experiment and is only rarely considered.…”

No Specific Subcellular Localization of Protein Kinase C Is Required for Cytotoxic T Cell Granule Exocytosis

“…However, there appear to be problems with the antibody we used. For example, it has been reported that it only detects an unphosphorylated form of PKC␦, not the phosphorylated form capable of catalytic activation (18). In light of the new information provided by Ma et al, we decided to revisit the role of PKC␦ in lytic granule exocytosis.…”