2.3. Cell Cycle and Western Blot Analysis

Cells were seeded in 10 cm dishes (220,000 cells/mL) and treated with 50 μM MMS (Sigma Aldrich) the next day. Cells were harvested for cell cycle and western blot analysis by trypsinization 12 and 24 h after treatment. Cells for cell cycle analysis cells were fixed in ice-cold methanol, washed with PBS, RNAseA-treated (100 μg/mL in PBS, 37 °C, 30 min) and DNA stained with propidium iodide (50 μg/mL in PBS). DNA staining was quantified using a FACS Canto flow cytometer (BD-Life Science, Franklin Lakes, NJ, USA) and FlowJo software. Cells for western blot analysis were lysed in 3 × packed cell volume (PCV) M-PER Mammalian Protein Extraction Reagent (Thermo Scientific, Waltham, MA, USA), PIC2 (10 μL/mL buffer) and PIC 3 (10 μL/mL buffer) (Sigma-Aldrich, Saint Louis, MO, USA) and complete protease inhibitor (20 μL/mL buffer) (Roche diagnostics, Basel, Switzerland) and incubated for 1 h at 4 °C. 1 μL Omnicleave was added to 100 μL packed cell volume (PCV). The lysate was cleared by centrifugation for 10 min at 16,000× g, 4 °C. Samples were run on 4–12% Bis-Tris-HCl (NuPAGE) gels. After blotting, the membrane (Immobilon PVDF, 0.2 μM) was blocked in 5% low fat dry milk in TBS (TBS with 0.1% Tween 20). The primary antibodies, CHK2-P (Thr68) (Cell signaling, 21975), CHK2 (Cell signaling, 3340), MCM2-P (S139) (Cell signaling, 8861), beta actin (Abcam, 8226), γH2AX (pSer319) (Biolegend), SHPRH (Abcam 80129), HLTF (Abcam 17984), P53-phospho ({"type":"entrez-protein","attrs":{"text":"CST92845","term_id":"904281042","term_text":"CST92845"}}CST92845), P53 (MA5-12571) as well as the secondary antibodies IRDye 800CW (Goat Anti-Rabbit) and IRDye 700RD (Goat Anti-Mouse) Secondary Antibody (LI-COR Bioscience, Lincoln, NE, USW) were diluted in 5% dry milk in TBS and the proteins were visualized using the Odyssey Imager.