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