Research Paper Volume 11, Issue 24 pp 12546—12567

Figure 2. p21 is deacetylated by Sirt1 in CMs. (A) P1 and P28 mouse heart lysates were immunoprecipitated with a p21 antibody and analyzed by Western blotting with Sirt1 antibody (n=5). (B) Sirt1 was precipitated from isolated P1 CMs with Sirt1 antibody and blotted with p21 antibody, and vice versa (n=5). Negative controls: IgG. (C) Isolated P1 CMs were transfected with Ad-NC or Ad-Sirt1. Whole cell lysates were immunoprecipitated with a p21 antibody and analyzed by Western blotting using an acetyl-lysine antibody; Western blotting was performed to assess Sirt1 and p21 protein expression in Ad-NC or Ad-Sirt1 P1 CMs. β-actin was used as a loading control (n=5). (D) Adult mouse hearts were injected with Ad-NC or Ad-Sirt1. Heart lysates were immunoprecipitated with a p21 antibody and analyzed by Western blotting with an acetyl-lysine antibody; Western blotting was performed to evaluate Sirt1 and p21 protein expression in Ad-NC or Ad-Sirt1 adult heart lysates (n=5). (E) Isolated P1 CMs were transfected with si-NC or si-Sirt1. Whole cell lysates were immunoprecipitated with a p21 antibody and analyzed by Western blotting using an acetyl-lysine antibody; Western blotting was performed to evaluate Sirt1 and p21 protein expression in si-NC or si-Sirt1 P1 CMs (n=5). (F) P1 mouse hearts were injected with Ad-si-NC or Ad-si-Sirt1. Heart lysates were immunoprecipitated with a p21 antibody and analyzed by Western blotting using an acetyl-lysine antibody; Western blotting was performed to evaluate Sirt1 and p21 protein expression in Ad-si-NC or Ad-si-Sirt1 adult heart lysates (n=5). Statistical significance was calculated using a two-tailed unpaired Student’s t-test in C–F. *p<0.05; data are presented as the mean ± S.E.M.