Fig. 6

TrkA promotes the ubiquitinated degradation of AGPS by MDM2 by modifying the phosphorylation of AGPS. a Western blot analysis of endogenous AGPS proteins in DU145 and 22Rv1 cells followed by treatment with different phosphatase inhibitors (Merestinib, 10 μM for 24 h; AG1295,10 μM for 2 h; AIM-100, 10 μM overnight; Dacomitinib, 2 μM for 24 h; Larotrectinib, 20 nM overnight). b Co-IP analysis of the phosphorylation levels of Flag-tagged AGPS in DU145 cells after being treated with MG132 (20 μM) for 8 h and Larotrectinib. c Western blot analysis of TrkA and p-TrkA expression in DU145, 22Rv1 and RWPE-1 cells. d Analysis of correlation between IHC staining of AGPS and p-TrkA proteins in different Gleason score PCa patient specimens. e Western blot analysis of AGPS protein expression in DU145 and 22Rv1 cells with or without Larotrectinib treatment. f Western blot analysis of ubiquitination levels in ectopically expressed Flag-tagged AGPS and ectopically expressed Myc-tagged MDM2 in DU145 cells treated with Larotrectinib. g Western blot analysis of ubiquitination levels in ectopically expressed Flag-tagged AGPS and ectopically expressed Myc-tagged MDM2 in DU145 cells treated with MG132 (20 μM) for 8 h or with Larotrectinib. h Western blot analysis of AGPS protein expression in DU145 cells with or without CHX (50 μg/mL) treatment for 0-, 4-, 8-, 12-, 16-, or 20- h. i Protein bands were quantified and normalized to the band intensity at the 0-h time point. * P < 0.05, ** P < 0.01, *** P < 0.001. j Co-IP analysis of binding of Flag-tagged AGPS with ectopically expressed Myc-tagged MDM2 in DU145 cells after NTRK1 knockdown. k Western blot analysis of ubiquitination levels in ectopically expressed Flag-tagged AGPS with ectopically expressed Myc-tagged MDM2 in DU145 cells after NTRK1 knockdown and treated with MG132 (20 μM) for 8 h