Ab2352

Total cell extracts were prepared from exponentially growing cells and were subjected to electrophoresis in 10% SDS-polyacrylamide gel and then transferred onto a PVDF membrane. The membrane was blocked with 5% skim milk. To detect XPA, APE1, or α-tubulin, the membrane was incubated in 1:100 dilution of anti-XPA monoclonal antibody (ab2352, Abcam), 1:2000 dilution of anti-APE1 monoclonal antibody (ab194, Abcam), or 1:10000 dilution of anti-α-tubulin monoclonal antibody (ab7291, Abcam) overnight. After washing with phosphate-buffered saline containing 0.05% Tween 20, the membrane was incubated with 1:2500 dilution of anti-mouse IgG conjugated to horseradish peroxidase (GE Healthcare Bio-Sciences). To detect OGG1 or DNA polymerase β (Pol β), the membrane was incubated with 1:10000 dilution of anti-OGG1 monoclonal antibody (ab124741, Abcam) or 1:1000 dilution of anti-Pol β polyclonal antibody (ab26343, Abcam) overnight. After washing with phosphate-buffered saline containing 0.05% Tween 20, the membrane was incubated with 1:2500 dilution of anti-rabbit IgG conjugated to horseradish peroxidase (GE Healthcare Bio-Sciences). The proteins were visualized by chemiluminescence using the ECL system (GE Healthcare Bio-Sciences).

γH2AX signal in HeLa cells by FACS was measured using a commercial kit (FlowCellect Multi-Color DNA Damage Response Kit, Millipore, Germany) following the manufacturer's protocol. The methods for the analyses of yeast UV survival curves, cell cycle profiles, Pulsed-Field Gel Electrophoresis and spontaneous and UV-induced Rad52 foci have been previously described in detail [15] (link). Cell treatment, protein extraction and Western Blotting for Rad53 activation has been performed strictly as previously described [23] (link). Rad53 antibody used was previously described [38] (link). For XPD and XPA Western blots, 25 µg of total amount of protein, extracted from U2OS cells, were used. Antibodies XPD (abcam ab54676, 1∶5000), XPA (abcam ab2352, 1∶1000) and β-Actin (abcam ab8226, 1∶5000) diluted in TBS-Tween 0.1% with 5% milk were incubated overnight at 4°C.