Sds page denaturing gels

For total lysates, cells were disrupted in lysis buffer (50 mM Tris–HCl, 20 mM NaCl, 1 mM MgCl₂, and 0.1% SDS) containing a protease and phosphatase inhibitor cocktail (Roche) supplemented with 0.1% endonuclease (benzonase, Millipore) for 10 min at room temperature with rotation. For fractionation analysis, cells were lysed using an NE-PER kit (ThermoFisher) following the manufacturer’s instructions. Laemmli buffer containing beta-mercaptoethanol was added to the samples, which were subsequently boiled for 5 min at 95 °C. The proteins were separated on SDS–PAGE denaturing gels (Bio-Rad) and transferred to nitrocellulose membranes (Bio-Rad). Next, the membranes were blocked with phosphate-buffered saline (PBS)–milk (5%) or PBS–bovine serum albumin (BSA) (3%) for 1 h, and signals were visualized using WesternBright ECL (Advansta) on a digital imaging system (GeneGnome, Syngene) or using Amersham Hyperfilm ECL film (GE) on a table-top processor (Curix 60, AGFA). The antibodies used in this study are listed in Supplementary Table S1.

Cells were collected by trypsinization, washed with cold PBS, re-suspended in SDS–polyacrylamide gel electrophoresis loading buffer (0.05 M Tris–HCl pH 6.8, 2% SDS, 10% glycerol, 0.01% bromophenol blue, 50 mM DTT) and passed through a needle and heated at 95 °C for 10 min. Samples were separated on SDS-PAGE denaturing gels (Bio-Rad) and transferred to nitrocellulose membranes (Bio-Rad). Membranes were blocked with PBS-Milk (5%) for 1 h before being incubated in the indicated primary antibody and the corresponding secondary antibody. Signals were visualized with WesternBright ECL (Advansta) on a digital imaging system (GeneGnome, Syngene) or using Amersham Imager 600 (GE).
Antibodies used: Vinculin (Abcam ab18058), FANCD2 (Abcam ab108928), SETX (Novus Biologicals NB100-57542), MUS81 (Abcam ab14387), XPF (Invitrogen MA5-12054).