Fig. 3

TRIM32 promotes STIM1 degradation via the ubiquitin-proteasome pathway in PCa. a Coimmunoprecipitation (Co-IP) analysis of interaction of STIM1 with TRIM32, MIB1 using anti-STIM1 antibody. b Co-IP analysis of interaction between TRIM32 and STIM1 using anti-TRIM32 antibody. c Co-IP analysis of interaction between exogenous STIM1 and exogenous TRIM32 in DU145 cells transfected with His-TRIM32 plasmid and Myc-STIM1 plasmid using anti-Myc antibody(up) or anti-His antibody (down). d-e Western blot (WB) analysis of STIM1 expression in TSPAN18-overexpressing (d) or TSPAN18-knockdown (e) DU145 and PC-3 cells. f the protein level of STIM1 in DU145 and PC-3 cells transfected with scramble or si-TRIM32 were monitored by WB at indicated times after cycloheximide (CHX, 20μg/mL), and were quantified by ImageJ software. The values are expressed as the mean ± s.d. of three independent experiments. *p<0.05, **p<0.01, ***p < 0.001, Student’s t test. g-h Co-IP analysis of ubiquitination of STIM1 in TRIM32-knockdown DU145 and PC-3 cells co-transfected with Myc-STIM1 plasmid and HA-Ub plasmid (g) or HA-Ub-K48 plasmid (h). i Co-IP analysis of interaction between exogenous TSPAN18 and exogenous TRIM32 in DU145 co-transfected with His-TRIM32 plasmid and Flag-TSPAN18 plasmid using anti-Flag antibody(upper) or anti-His antibody (lower). j-k HEK-293T cells were co-transfected with indicated plasmid for 48h, then Co-IP analysis was conducted with anti-STIM1 antibody. l-m WB analysis of whole-cell lysates (left) or IP (right) from cells co-expressing Myc-STIM1 CC1, Myc-STIM1 CAD, Myc-STIM1 CC1-CAD, or Myc-STIM1 CT and Flag-TSPAN18 (l) or His-TRIM32 (m) in HEK-293T cells