Imobilon p membrane

Immunoprecipitation was performed by incubating equal amounts of cell lysates with an anti-TopBP1 mouse monoclonal antibody or control mouse IgG and then with protein A/G agarose beads (from GenDepot) for 16 h at 4 °C. After three washes, immunoprecipitates were fractionated by SDS-PAGE and electrotransferred to Imobilon-P membrane (Millipore). Immunoblotting was performed with appropriate antibodies. Antibody specific to E2F1 (C-20 or KH-95), Chk-1 (G-4), Rad9 (M-389), or GAPDH (6C5) was from Santa Cruz Biotechnology. Anti-TopBP1 mouse monoclonal antibody was from BD Transduction Laboratories. Anti-TopBP1 (BL893) rabbit polyclonal antibody was from Bethyl Laboratories. Anti-phospho-TopBP1 (S1159) antibody was from Abgent. Antibody specific to p-Chk1(S345), ATR, ATRIP, BACH1/BRIP1, RPA32, or Histone H3 was from Cell Signaling.

Liver samples were homogenised in buffer consisting of 10 mM Tris–HCl pH 7.5, 1 mM EGTA, 75 mM sucrose, 225 mM sorbitol and 0.1% BSA, before lysis at 4°C in 50 mM Tris–HCl pH 7.4, 1 mM EDTA, 150 mM NaCl, 1% NP40, 0.1% SDS and 1 × protease inhibitor cocktail (Roche). Protein concentration was determined by Lowry assay (DC Reagent, Bio-Rad), and 5 μg of protein resolved on PAGE gels (Novex) before transfer to Imobilon-P membrane (Millipore). Following blocking with 5% non-fat dry milk in PBS with 0.1% (v/v) Tween-20, membranes were incubated overnight with the following primary antibodies: mouse anti-NDUFB8 (1:10 000, Abcam AB110242), mouse anti-COX IV (1:10 000, Abcam AB14744), rabbit anti-MPV17 (1:1000, Proteintech 10310-1-AP) or rabbit anti-TOM20 (1:20 000, Santa Cruz Biotechnology SC-11415). Membranes were incubated with HRP-conjugated anti-mouse or anti-rabbit secondary antibody (1:4000, Promega) in 5% non-fat dry milk in PBS with 0.1% (v/v) Tween-20, and visualised using enhanced chemiluminescence (ECL, G.E. Healthcare).