Cell lytic m lysis reagent

To prepare whole cell lysates, cells were lysed with CellLytic^™M lysis reagent (C2978, Sigma-Aldrich). After thorough mixing and incubation at 4°C for 30 min, lysates were centrifuged at 15,000 g at 4°C for 10 min, and supernatants were collected. To prepare chromatin bound subcellular fraction, we followed the protocol of Subcellular Protein Fractionation Kit from Thermo Scientific (78840). Samples were mixed with tris-glycine SDS sample buffer (Novex, LC2676) and loaded onto Novex tris-glycine gels (Novex). Blotted membrane was blocked with 5% bovine serum albumin (BSA) (Sigma-Aldrich. A9418) in phosphate-buffered saline (PBS) with 0.1% tween-20 (PBST). The primary antibodies were diluted in 5% BSA/PBST by 1:3000 for phospo-S345-CHK1, CHK1, phspho-S4/S8-RPA2, RPA1, CtIP, CDC45, DHX9, cyclin A and ORC2, and 1:10000 for phospho-S139-H2AX, SLFN11 (D-2), RPA2, Histone H3, GAPDH, PCNA, MCM3 and MCM2. The secondary antibodies were diluted in 5% non-fat milk by 1:10000. Quantification of band intensity was done using ImageJ software. A proper size of square that was slightly larger than blot bands was set, and used to measure the mean intensity of each band. The square size was consistent through the experiment for each antibody. Intensity of background was subtracted, and the intensity of each control sample was set as 1.