Fig. 7: Lysine 451 mutant SMAD2 decreased the ubiquitization induced by SMURF2 and the acetylation of SMAD2 induced by CBP and P/CAF.

a, b HK2 cells transfected with Ad-SIRT2 and/or siSMURF2 for 24 h (Ad-Null and siCtrl were used as controls, respectively), and treated with 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 (a), with the quantitative results shown in b (n = 3). c,d HK2 cells were 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 subjected to Co-IP with anti-SMAD2 antibody, followed by western blotting using indicated antibodies (c), with quantitative results shown in the (d) (n = 3). e, f HEK293T cells were transfected with Ad-WT SMAD2, Ad-mutant SMAD2, or Ad-SIRT2 as indicated for 24 h, and treated with 2 ng/ml TGF-β for 6 h. HEK293T cell lysates subjected to Co-IP with anti-SMAD2 antibody, followed by western blotting using indicated antibodies (f). Quantitative results are shown in the right panel (n = 3). g SMURF2-SMAD2 K451 docking with the HDOCK server. High magnification of boxed areas is presented on the left. Arrow indicates SMAD2 protein K451 site. h CBP-SMAD2 K451 docking with the HDOCK server. High magnification of boxed areas is presented on the right. Arrow indicates SMAD2 protein K451 site. i HEK239T cells were transfected with lentivirus-CBP or lentivirus empty vector for 48 h, and subjected to Ad-WT-SMAD2 or Ad-Mut-SMAD2 transfection as indicated for 24 h. HEK293T cell lysates subjected to Co-IP with anti-SMAD2 antibody, and western blotting using indicated antibodies. Quantitative results are shown in the right panel (n = 3). j P/CAF-SMAD2 K451 docking with the HDOCK server. High magnification of boxed areas is presented on the left. Arrow indicates SMAD2 protein K451 site. k HEK239T cells were transfected with Ad-WT-SMAD2, Ad-Mut-SMAD2, or Ad-P/CAF as indicated for 24 h. HEK293T cell lysates subjected to Co-IP with anti-SMAD2 antibody, and western blotting using indicated antibodies. Quantitative results were shown in the right panel (n = 3). Key in (b) also applies to (d, g). Key in (i) also applies to (k). 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.