Mouse monoclonal anti pcna

WT and Ercc1^−/− immortalized MEFs at 60% confluence were treated with 20 μM HNE in serum-free medium in the presence of 15 μM ML323, an inhibitor of PCNA deubiquination (Axon Medchem, Groningen, The Netherlands). After 2 h, the treatment medium was replaced with a fresh growth medium with 15 μM ML323. Cells were harvested by scraping at 2, 6, 10 h following HNE exposure and 10 h for mock-treated (the longest time of incubation with inhibitor). Simultaneously, UVC-irradiated fibroblasts (60 J/m²) were prepared as a positive control. Cells were cultured for 6 h after exposure to UV light in a growth medium containing 15 μM ML323 and subsequently collected. To prepare chromatin fractions, cells were resuspended in RIPA buffer (Sigma-Aldrich) supplemented with protease inhibitor cocktail (Complete, Roche) and 0.25 mM PMSF (Sigma-Aldrich). The cell suspensions were incubated on ice for 30 min and centrifuged at 15000×g for 15 min. The pellet was collected and washed with RIPA buffer three times followed by adding 2× Laemmli buffer, sonication and boiling. The final solution was used as the chromatin fraction. Western blot analysis was performed using standard protocols. Proteins were separated on 10% SDS-PAGE. The primary antibodies used were mouse monoclonal anti-PCNA (Santa Cruz Biotechnology) and rabbit polyclonal anti-lamin A/C (Santa Cruz Biotechnology).

For harvested tumors or cell lines, whole cell protein isolation, hydrochloric acid extraction of histones, western blotting and signal detection were performed as previously described [23 (link), 24 (link)]. Antibodies used were rabbit polyclonal anti-BRD4 (Millipore), mouse monoclonal anti-BRCA1 (Millipore), rabbit polyclonal anti-RAD51 (Millipore), rabbit polyclonal anti-cyclin E (Abcam, Cambridge, MA), rabbit polyclonal anti-PARP (Cell Signaling Technology), rabbit polyclonal anti-Bax (Millipore), mouse monoclonal anti-PCNA (Santa Cruz Biotechnology, Inc., Dallas, TX), mouse monoclonal anti-p21 (Thermo Fisher), mouse monoclonal anti-phospho H2AX (Ser139) (Millipore), phospho(T2609)-DNA-PKcs (Thermo Scientific), DNA-PKcs (Santa Cruz Biotechnology, Dallas, TX). Loading control was β-actin (Sigma).

Whole cell protein isolation from cultured cells and harvested tumors, hydrochloric acid extraction of histones, western blotting and signal detection were as described [19 (link)]. Antibodies used were rabbit polyclonal anti-cyclin E (Abcam, Cambridge, MA), rabbit polyclonal anti-E2F1 (DBA Acris Antibodies, Inc, Rockville, MD), rabbit polyclonal anti-RAD51 (Millipore), mouse monoclonal anti-BRCA1 (Millipore), rabbit polyclonal anti-PARP (Cell Signaling Technology), mouse monoclonal anti-PCNA (Santa Cruz Biotechnology, Inc., Dallas, TX), rabbit polyclonal anti-cleaved caspase-3 (Cell Signaling Technology), and mouse monoclonal anti-pH2AX (Ser139) (Millipore). Loading controls were mouse monoclonal anti-histone H3 (Millipore) and mouse monoclonal β-actin (Sigma) for histones and total proteins, respectively.