Rabbit anti phospho chk1 ser317

In order to detect MCPH1 expression, cell or tissue samples were lysed with RIPA buffer supplements with protease/phosphatase inhibitors (APExBIO). An amount of 40–60 ug total protein was separated with SDS-PAGE gels (10%), and further transferred onto PVDF membranes. The primary antibodies used for this study were the following: rabbit anti-MCPH1 (1:1000, Cell Signaling, Danvers, MA, USA); and mouse anti-β-actin (1:10,000, Sigma-Aldrich, St. Louis, MO, USA). For the DNA damage assay, MEFs were treated with hydroxyurea (HU, 2 mM) for 3 h, and then recovered for 0, 3 and 6 h. Protein samples were harvested at different time-points. In order to investigate the DNA repair dynamics, the following antibodies were used: mouse anti-phospho histone H2A.X (Ser139) (1:2000, 05-636, EMD Millipore, Burlington, MA, USA), rabbit anti-phospho Chk1 (Ser317) (1:1000,12302S, Cell Signaling, Danvers, MA, USA) and mouse anti-β-actin (1:10,000, A5441, Sigma-Aldrich, St. Louis, MO, USA). The secondary antibodies used were: HRP-conjugated goat anti-rabbit IgG and HRP-conjugated goat anti-mouse IgG (1:2000; Proteintech, Tokyo, Japan). The blotting result was developed using BeyoECL Plus substrates (Beyotime, P0018S, Shanghai, China) and quantified with Evolution-Capt Solo S 17.00 software. The antibodies used for WB are summarized in supplementary files (Supplementary Tables S2 and S3).