Fig. 4

JA2131 induces hyperPARylation of PARP1. a Subcellular PARG protein expression patterns in cultured cells. Subcellular fractionated lysates were immunoblotted with anti-PARG, (upper panel) followed by nuclear (N) marker anti- Laminin Subunit Beta 1 (LAMB1, upper middle panel), chromatin (Chr) marker anti-Histone H3 (H3, lower middle panel) and the cytoplasmic (C) marker anti- Glyceraldehyde 3-phosphate dehydrogenase (GAPDH, lower panel) b PC3 cells were treated with DMSO or PARG inhibitor JA2131 (2131) for 2 h then irradiated with 7 Gy and allowed to recover for 0.5 h or 1.0 h before lysis and subcellular fractionation. Chromatin bound cell-extracts were analyzed with anti-PARP1 antibody (upper panel) followed by probing with total Histone H2AX (t-H2AX, lower panel) as the loading control. c DMSO or 2131-treated PC3 cells were irradiated and recovered for 2 h as above, chromatin fractions were then incubated with or without purified PARG enzyme (±PARG) for 30 min at 37 °C then immunoblotted with anti-PARP1 (upper panel) and then with anti-histone H3 (lower panel) as loading control. d Quantitative analysis of western blot of b, where expression of PARP1 levels in DMSO treated was normalized to 1. High molecular weight (HMW) in red PARP1 is significantly decreased in the presence of purified truncated PARG protein. The low molecular weight in black PARP1 bands are not significantly affected, n = 3. Plus denotes added purified PARG enzyme. Source Data are provided as a Source Data file.