Ectopic over-expression of PARG influences histone acetylation levels.

(A) Representative Western blot of total proteins from cells transfected with empty vector (pCS2) or Myc–catalytically mutated PARG construct (pCS2-Myc-PARG_E757N) or Myc–PARG construct (pCS2-Myc-PARG) run on an 8% SDS-PAGE, and probed with anti-Myc epitope to check for exogenous protein expression. (B) Same as in (A) but hybridisation was performed with anti-PAR antibody to visualize PARs level, and with anti-α Tubulin as protein loading control. (C) The same extracts used in panel A were run on a 15% SDS-PAGE, and probed with anti-acetyl-histone H3 and anti-C-terminal of H3 antibodies. Histograms indicate acetylation level of cells transfected with pCS2-Myc-PARG construct (black), or with pCS2-Myc-PARG_E757N (grey) relative to control cells transfected with empty vector (white, value ~1). Error bars indicate the standard deviation of data obtained from three independent experiments. (D) As in (C), but using anti-acetyl-histone H4 and anti-C-terminal of H4 antibodies to measure the relative level of H4 acetylation. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001(A) 转染空载体（pCS2）、Myc标记的催化突变型PARG构建体（pCS2-Myc-PARG_E757N）或Myc标记的PARG构建体（pCS2-Myc-PARG）的细胞总蛋白，经8%十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分离后，采用抗Myc表位抗体进行免疫印迹检测，以验证外源蛋白的表达情况，图中为代表性检测结果。(B) 实验设置与(A)一致，但分别使用抗PAR（poly(ADP-ribose)，多聚ADP核糖）抗体检测PAR水平，以及抗α微管蛋白（α-Tubulin）抗体作为蛋白上样量对照进行杂交。(C) 使用与(A)组相同的细胞提取样品，经15% SDS-PAGE分离后，分别采用抗乙酰化组蛋白H3抗体与抗H3羧基端抗体进行检测。直方图展示了转染pCS2-Myc-PARG构建体（黑色柱）、pCS2-Myc-PARG_E757N（灰色柱）的细胞的组蛋白H3乙酰化水平，以转染空载体的对照细胞为基准（白色柱，数值约为1）。误差棒表示三次独立实验所得数据的标准偏差。(D) 实验设置与(C)一致，但分别使用抗乙酰化组蛋白H4抗体与抗H4羧基端抗体，以检测组蛋白H4的相对乙酰化水平。*p ≤ 0.05；**p ≤ 0.01；***p ≤ 0.001