Fig. 6: SIRT2 regulated TGF-β signaling by deacetylating lysine 451 on SMAD2.

a SIRT2-SMAD2 K451 docking with the HDOCK server. High magnification of boxed areas is presented on the right. Left arrow indicates the SMAD2 protein 447-456 peptide; right arrow indicates SMAD2 protein K451 site. b The conservation of SMAD2 lysine 451 in different species. c, d HEK293T cells transfected with Ad-WT SMAD2, Ad-mutant SMAD2, or Ad-SIRT2 for 24 h, followed by the treatment of 2 ng/ml TGF-β for 6 h. HEK293T cell lysates were subjected to Co-IP with anti-SMAD2 antibody, followed by western blotting using indicated antibodies, and the quantitative results are shown in the right panel (n = 3). d HEK293T cells were transfected with Ad-WT SMAD2 or Ad-mutant SMAD2 for 24 h, followed by the treatment of with or without 2 ng/ml TGF-β for 6 h. Western blot analyses of p-SMAD2 (Ser465), SMAD2, and β-actin in the whole cells lysates or fractions extracted from HEK293T cells, with quantitative results shown in the right panel (n = 3). e HEK293T cells transfected with Ad-WT SMAD2, Ad-mutant SMAD2 for 24 h, followed by the treatment with or without 2 ng/ml TGF-β for 2 h. Representative images of SIRT2 immunofluorescence staining are shown in the HEK293T cells (scale bar = 50 μm). f, g HEK239T cells were transfected with Ad-WT SMAD2, Ad-mutant SMAD2, or Ad-SIRT2 as indicated for 24 h, followed by the treatment with 2 ng/ml TGF-β for 6 h. g Western blot analyses of p-SMAD2 (Ser465), SMAD2, and β-actin in the whole cells lysates or fractions extracted from HEK293T cells, and the quantitative results are shown in the right panel (n = 3). h Representative images of SIRT2 immunofluorescence staining are shown in the HEK293T cells (scale bar = 50 μm). Key in (c) also applies to (d–g). For all panels, data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA with a Bonferroni correction test.