Figure 1

SUMOylation of aPKC.

(A) Lysates prepared from HEK293T cells co-expressing HA-PKCζ, Myc-Par6 or HA-Par3 with GFP-SUMO1G or GFP-SUMO1GG were subjected to immunoprecipitation (IP) using GFP-specific antibodies and the immunoprecipitates were analyzed for the presence of specific proteins by western blotting (WB) using indicated antibodies. *indicates SUMO modified bands. Note that SUMOylated bands could be detected in the cases of HA-PKCζ and Myc-Par6, not in case of HA-Par3. (B) Cells co-expressing HA-PKCζ and GFP-SUMO2G or GFP-SUMO2GG were immunoprecipitated with GFP antibodies and probed for the presence of PKCζ using HA-specific antibodies. *indicates SUMO modified band. (C) HEK293T cells were lysed and IP was performed with control mouse IgG (IgG-IP) or mouse anti-aPKC (aPKC-IP) antibodies and the immunoprecipitates were subjected to western analysis using SUMO1 or SUMO2/3 antibodies. (D) SUMOylation of recombinant PKCζ was performed using in vitro SUMOylation kit as per manufacturer’s instruction. The products were analyzed by western blotting using indicated antibodies (E) HEK293T cells were lysed and subjected to IP using mouse IgG (IgG-IP) or mouse anti-aPKC antibodies (aPKC-IP) and western analysis was performed to monitor the presence of Ubc9.