5C). In addition, IP experiments using anti-phosphorylated http://www.selleckchem.com/products/AG-014699.html PKC antibody (9379) confirmed that knocking down BART inhibited binding of ANX7 and phosphorylated PKC (Fig. 5D, E). Figure 5 Effect of BART on regulating PKC activity through ANX7. BART RNAi S2-013 cells had elevated active PKC levels and unchanged steady state levels of PKCs (Fig. 5F). This result indicates that BART may be associated with decreased levels of active PKC. We hypothesize that BART regulates interactions between ANX7 and active forms of target PKCs as a scaffold molecule and/or a cargo protein of ANX7, allows ANX7 to decrease target PKC activity, and in turn, inhibits cell invasion.

PKC activity is not directly regulated by ANX7 To investigate the role of PKC in regulating phosphorylation of ANX7, as previously reported in chromaffin cells [8], S2-013 and PANC-1 cells were metabolically labeled with [32P]-orthophosphoric acid, and then stimulated with PMA. The radioactively labeled-ANX7 was immunoprecipitated with anti-ANX7 monoclonal antibody and was analyzed by phosphor imaging (Fig. 6A). If ANX7 is a substrate of specific PKCs, the level of ANX7 phosphorylation should result in significantly increased changes in response to PMA. However, PMA stimulation did not increase the levels of ANX7 phosphorylation in either cell line. Next, in vitro phosphorylation assays were used to determine whether PKC activity was directly regulated by ANX7 (Fig. 6B). Purified rat brain, with a purity of >95% and containing classical and novel PKC isoforms, was incubated with recombinant ANX7 protein with or without recombinant BART protein.

ANX7 did not change the activity of PKCs and adding BART protein was not associated with regulating PKC activity. These results suggest that ANX7 is not a substrate of PKC, and that ANX7 does not change phosphorylation levels of the target PKCs directly. Figure 6 PKC phosphorylation is not directly regulated by ANX7. PKC�� is associated with BART-ANX7 complexes To identify specific isoforms of PKC that bind to ANX7 in this system, a precise expression profile of classical and novel PKCs was generated by Western blotting using individual anti-phospho-PKC antibodies in BART RNAi cells derived from S2-013 (Fig. 7A). Since phosphorylation levels of target PKCs were increased in BART RNAi cells (Fig. 5F), upregulated phospho-PKCs in BART RNAi cells were selected for further analysis.

Among these, PKC�� was significantly activated by BART knockdown in S2-013. In addition, PKC�� was abundantly GSK-3 phosphorylated in ANX7 RNAi cells of S2-013 and PANC-1 (Fig. 7B). Next, binding of ANX7 with phosphorylated PKC�� was demonstrated by immunoprecipitation and Western blotting analysis in S2-013 cells (Fig. 8A). Phospho-PKC�� was not immunoprecipitated with ANX7. To investigate the subcellular colocalization of phosphorylated PKC��, S2-013 cells were immunostained.