Figure 5: PKC is involved in regulating Gcm and GcmR59L phosphorylation.
(a,b) Co-IP analysis showed that both Gcm and GcmR59L interact with PKC, albeit with a different efficiency. Inputs were shown in (b). Asterisk on the right side indicated non-specific background bands. (c) Gcm expressing S2 cell lysates were treated with OA, PMA, or co-transfected with PKC, pulled down with the anti-Flag antibody, and analyzed with the anti-p-Ser-PKC antibody (top panel), the anti-Flag antibody, (second panel), and the anti-HA antibody (third panel). Inputs for each column were shown in the bottom three panels. Asterisk on the right side indicated 3xFlag-Gcm. Note an increase in the signal intensities when Gcm was co-transfected with PKC or treated with the PKC activator PMA. (d) S2 cell lysates transfecting Gcm or GcmR59L and PKC were analyzed by 10% SDS-PAGE gel. Note the intensities for the upper slower migrating band representing phosphorylated Gcm or GcmR59L were enhanced upon PKC co-transfection and more phosphorylated GcmR59L (the upper slower migrating band) were detected compared to Gcm, both in the presence of PKC. (e) S2 cell lysates transfected with Gcm or GcmR59L were pulled down by the anti-Flag antibody, treated with CIP, subsequently analyzed by SDS-PAGE. Note that two bands designated with asterisks represented phosphorylated Gcm (P-Gcm) and dephosphorylated Gcm (Gcm). GcmR59L exhibited a higher phosphorylation level than Gcm upon CIP treatment. Western blot gels have been run under the same experimental conditions.
