Fig. 5: SIRT2 interacts with SMAD2 and promoted its degradation.
From: SIRT2 alleviated renal fibrosis by deacetylating SMAD2 and SMAD3 in renal tubular epithelial cells
a, b HK2 cell lysates were subjected to co-immunoprecipitation (Co-IP) with anti-SIRT2 or anti-SMAD2 antibody, respectively. Normal rabbit IgG was used as the control, and western blotting was conducted using the indicated antibodies. c HK2 cells transfected with Ad-Null or Ad-SIRT2 for 24 h, and treated with or without 2 ng/ml TGF-β for 6 h. HK2 cell lysates were subjected to Co-IP with anti-SMAD2 antibody, followed by western blotting using the indicated antibodies. The quantitative results are shown in the right panel (n = 3). d HK2 cells transfected with Ad-Null or Ad-SIRT2 for 24 h, and treated with or without protease inhibitor MG132 (20 μM) in the present of TGF-β for 8 h. Western blot analysis of SIRT2, SMAD2, and β-actin in HK2 cells, with the quantitative results shown in the right panel (n = 3). e HK2 cells were transfected with Ad-Null or Ad-SIRT2, and treated with CHX under TGF-β stimulation for indicated times. Western blot analysis of SIRT2, SMAD2, and β-actin in HK2 cells, with the quantitative results shown in the right panel (n = 3). f, g PTECs isolated from Sirt2−/− mice and were transfected with Ad-Sirt2, Ad-Sirt2-N168A, and Ad-Sirt2-H187Y for 24 h, followed by 6 h TGF-β stimulation. PTECs cell lysates subjected to Co-IP with anti-SMAD2 antibody, followed by western blotting using indicated antibodies, and the quantitative results were shown in the right panel (n = 3). h,i SIRT2 siRNA (siSIRT2) or Ctrl siRNA transfected with HK2 cells for 24 h, followed by the treatment with/without 2 ng/ml TGF-β for 6 h. HK2 cell lysates were subjected to Co-IP with anti-SMAD2 antibody, followed by western blotting using indicated antibodies, and the quantitative results were shown in the bottom panel (n = 3). j HK2 cells transfected with Ctrl siRNA or siSIRT2 for 24 h, followed by treated with or without protease inhibitor MG132 (20 μM) in the present of TGF-β for 8 h. Western blot analysis of SIRT2, SMAD2, and β-actin in HK2 cells, and the quantitative results were shown in the right panel (n = 3). The key in (a) also applies to (b–f); the key in (g) also applies to (h). For all panels, data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 by a one-way ANOVA with a Bonferroni correction test.
