Figure 4

Nup358 acts as E3 ligase for aPKC SUMOylation.

(A) HEK293T cells were transfected with control (siControl) or Nup358 specific (siNup358) siRNA and were lysed and analyzed for the levels of indicated proteins using specific antibodies by western blotting (WB). *indicates SUMO-modified band. Extent of Nup358 depletion was monitored with Nup358 specific antibodies. Vinculin was used as loading control. Numbers denote relative intensities of indicated bands. (B) siRNA treated cells were subjected to nucleo-cytoplasmic fractionation and presence of aPKC in the nuclear and cytoplasmic fractions was assessed by WB. Lamin A/C and Vinculin were used as markers for nuclear and cytoplasmic fractions, respectively. The graph represents quantitative data for relative aPKC nucleo-cytoplasmic distribution under control and Nup358 depleted conditions. Error bars indicate standard deviations, n = 3, P values calculated by Student’s t test. The extent of Nup358 depletion was monitored by western blotting with specific antibodies and Vinculin was used as loading control. Numbers denote relative intensities of indicated bands. (C) Cells were initially transfected with siControl or siNup358 and were retransfected with FLAG-Lgl1 with (+) or without (−) HA-PKCζ. Cell lysates were analyzed by WB using indicated antibodies. Numbers denote relative intensities of indicated bands. (D) Lysates prepared from HEK293T cells transfected with siControl or siNup358 were analyzed for the levels of indicated proteins. Vinculin was used as loading control. Graphs represent quantitative data for relative levels of phosphorylated p-MARK2 as compared to total MARK3. Error bars indicate standard deviations, n = 3, P values calculated by Student’s t test. (E) Schematic depiction of C-terminal region of human Nup358 (Nup358-C) with amino acids marked in number. Nup358-C contains two RanGTP binding domians (RB3 and RB4), internal repeat (IR) that acts as SUMO E3 ligase and cyclophilin homology domain (CHD). Dashed line shows the deleted region in Nup358-C mutant (Nup358-CΔIR). (F) HEK293T cells were transfected with GFP-control or GFP-Nup358-C construct and monitored for SUMOylation of endogenous aPKC using specific antibodies by WB. *indicates SUMOylated aPKC species. (G) Cells were co-transfected with indicated constructs and lysates were immunoblotted for indicated proteins. Graph represents quantitative data depicting the relative p-Lgl1 levels compared to total Lgl1 levels. Error bars indicate standard deviations, n = 3, P values calculated by Student’s t test.