Figure 2

Identification of SUMOylation sites in PKCζ.

(A) Schematic diagram of PKCζ depicting the domain architecture: PB1 (Phox and Bem 1), ZnF (zinc-finger) and kinase domains. Potential lysine (K) residues that were predicted by SUMOplot software as SUMOylation sites are marked with asterisks. (B) HEK293T cells were transfected with GFP-SUMO1G or GFP-SUMO1GG along with HA-empty vector control, HA-PKCζ-wt, HA-PKCζ lysine (K) to arginine (R) single mutants (K225R, K284R, K378R), double mutants (K225/284R, K284/378R, K378/225R) or triple mutant (K225/284/378R). Cells were lysed 36 h post-transfection and co-IP was performed using anti-HA antibodies, followed by western blot analysis with indicated antibodies. For better viewing of unmodified and SUMO-modified PKCζ, western blots corresponding to different exposures are provided (HA-IP and HA-WB panels). The graph represents quantitative data depicting levels of SUMOylated aPKC relative to unmodified PKCζ in the corresponding immunoprecipitates. Error bars indicate standard deviations, n = 3, P values were calculated by one-way ANOVA. P value is considered statistically significant if it is less than the critical level and is denoted by *. N.S. indicates non-significant.