Figure 5. Regulation of TGF-β1 by recruiting Pin1 into PML-NBs during HERG protein degradation.

(A) Co-immunoprecipitation analysis of PML interaction with Pin1 in cultured NMCMs. Total lysates immunoprecipitated with anti-Pin1 and anti-PML antibody were detected with anti-PML and anti-Pin1 antibodies, respectively. (B) Representative immunofluorescent staining of NMCMs immunostained with specific antibodies against PML (green) and Pin1 (red). (C) Immunofluorescence analysis performed using anti-PML (green) and anti-Pin1 (red) antibodies in NMCMs exposed to ATO (2 μM) for 2 h or Ang II (100 nM) for 4 h and transfected with the indicated siRNAs. Figure B–C: scale bar, 20 μm. An enlarged view of a single PML-NB in the boxed region is shown in the right panel. The representative images of three separate experiments are shown. Immunoblot analysis of TGF-β1 and GAPDH expression in UBC9-silenced NMCMs treated with (D) ATO (2 μM) or (E) Ang II (100 nM) for 24 h. TGF-β1 and GAPDH expression was analyzed by immunoblot of whole lysates from RNF4-silenced NMCMs treated with (F) ATO (2 μM) for or (G) Ang II (100 nM) for 24 h. The protein molecular masses are in kDa and are shown in the right panel. The high molecular-weight bands (> 130 kDa) in parentheses are SUMOylated PML (S-PML). The data shown are representative of three separate experiments.