Research Paper Volume 11, Issue 24 pp 12546—12567

Figure 1. Deacetylation of p21 attenuates p21 stability and CM cell cycle arrest. (A) P1 and P28 mouse heart lysates were immunoprecipitated with a p21 antibody and analyzed by Western blotting with acetyl-lysine antibody; Western blotting was used to evaluate p21 protein expression in P1 and P28 mouse heart lysates. β-actin was used as a loading control (n=5). (B) Isolated P1 CMs were transfected with NC, p21-WT, p21-KR, or p21-KQ. EdU incorporation was detected by immunofluorescence. Scale bar, 50 μm. Quantitative analyses represent fields from 5 mice per group. (C) Isolated P1 CMs were transfected with NC, p21-WT, p21-KR, or p21-KQ and quantification of counts from P1 CMs transfected with NC, p21-WT, p21-KR, or p21-KQ (n=5). cTnT immunofluorescence was assessed to detect CM numbers. Scale bar, 500 μm. (D) Isolated P1 CMs were transfected with NC, FLAG-p21-WT, FLAG-p21-KR or FLAG-p21-KQ. Whole cell lysates were immunoprecipitated with a p21 antibody and analyzed by Western blotting with an ubiquitin antibodies; Western blotting was used to detect p21 levels. β-actin was used as a loading control (n=5). Statistical significance was calculated using a two-tailed unpaired Student’s t-test in (A) and a one-way ANOVA followed by an LSD post hoc test in (B–D). *p<0.05; data are presented as the mean ± S.E.M.