Fig. 7

Pro enhances the inhibitory binding of 14–3-3ζ to C-Raf and blocks the positive feedback loop of TGF-β1 in OB-educated PCa. A Immunoprecipitation of 14–3-3ζ by C-Raf in C4-2B cells treated with DMSO or Pro (400 nM). IgG was used as the negative control. B Representative IF images of subcellular location of 14–3-3ζ and C-Raf in C4-2B cells treated with DMSO or Pro (400 nM). Green and red arrows indicate the separate locations of 14–3-3ζ and C-Raf; yellow arrows indicate the co-localization of 14–3-3ζ and C-Raf. C Western blot of the phosphorylation level of proteins in C-Raf/MAPK pathway in C4-2B cells treated with DMSO or Pro (400 nM). D Western blot of the phosphorylation level of indicated proteins in co-cultured C4-2B cells treated with DMSO, SB-431542 (10 μM) or Pro (400 nM). E Western blot of TGF-β1 in co-cultured C4-2B cells treated with DMSO, PD98059 (10 μM) or Pro (400 nM). F Fold change of TGF-β1 mRNA in C4-2B cells cultured alone and co-cultured with MC3T3 and treated with TGF-β1 (10 ng/ml) and Pro (400 nM). G Western blot of the phosphorylation level of proteins in C-Raf/MAPK pathway with or without 14–3-3ζ knockdown under indicated conditions. H Western blot of TGF-β1 in C4-2B-sh140 cell treated with DMSO, PD98059 (10 μM) or Pro (400 nM). I IHC staining of 14–3-3ζ and TGF-β1 in serial bone sections. J Schematic of Pro binding to 14–3-3ζ and blocking the positive feedback loop of TGF-β. ***P < 0.001