Fig. 7: TM4SF5-mediated complex formation with NCOA3 and PTEN led to NCOA3 stabilization and PTEN inactivation.
a Huh7KO cell variants in normal culture condition with 10% FBS were were collected for whole-cell lysates (WCL), before co-immunoprecipitation (IP) using anti-TM4SF5EC2 antibody. b Huh7KO cell variants were replenished with nutrients as indicated for 15 h and collected before immunoblots for the indicated molecules. c–g Huh7KO cell variants were or were not replenished with ALB to GLU + SFM for 0, 10, 30 or 60 min with or without MG132 (10 μM) treatment to block NCOA3 degradation: WCL were processed by IP using normal IgG (IgG), anti-TM4SF5EC2 polyclonal antibody (c and d, before immunoblotting for NCOA3 and either TM4SF5EC2 (c) or ubiquitin (d)), anti-NCOA3 rabbit monoclonal antibody (e), strep (f) or anti-PTEN rabbit monoclonal antibody (g). Immunoprecipitates were then immunoblotted in parallel to WCL and in some cases, cells were transfected with siNS or siNCOA3 for 24 h before nutrient replenishment (f). Ratio values of band intensities of certain immunoblots measured by using ImageJ software were normalized to those of the loading control or the total form of the molecule are presented. h Huh7KO cell variants were transfected with siNS or siNCOA3 for 24 h. ALB replenishment to GLU + SFM was performed for 0 or 10 min before the PTEN activity assay. P values were calculated via one-way ANOVA or unpaired Student’s t-tests, and P < 0.05 was considered statistically significant. Data represent three independent experiments.
