Ab177946

Whole-cell lysates were prepared with RIPA buffer containing protease inhibitor (Sigma) and phosphatase inhibitor (Roche Applied Science) cocktail tablets and the protein concentration were determined by Bio-Rad Protein assay (Bio-Rad). Equivalent amounts of protein were resolved by SDS-PAGE and transferred to polyvinylidene fluoride microporous membrane (Millipore), blocked with 1.5% BSA in H20 containing 0.1% Tween-20 (TBS-T), and membranes were probed with the following primary antibodies: anti-LIG1 (Abcam ab177946) (1:1,000), anti-FXR1 (Abcam ab155124) (1:1,000), and anti-GAPDH (Thermo Scientific MA5-15738) (1:3,000). Secondary antibodies were conjugated to horseradish peroxidase (HRP) and peroxidase activity was visualized using Chemiluminescent HRP substrate (Thermo Scientific).

Rabbit polyclonal anti-BRCA2 (1:200), anti-RAD51 (1:1,000), anti-RAD52 (1:500), anti-RIF1 (1:200), and anti-Polθ (1:200) antibodies were generated by immunizing rabbits with GST-BRCA2 (residues 2800–3000), MBP-RAD51, GST-RAD52, MBP-RIF1 (residues 1367–1461), and MBP-Polθ (residues 2125–2320) fusion proteins expressed and purified from E. Coli, respectively. Anti-ERCC1 (1:2000, ab129267), anti-RPA2 (1:1000, ab2175), anti-Lig1 (1:2000, ab177946), anti-Lig4 (1:2000, ab193353) and anti-53BP1 (1:20,000, ab175188) antibodies were purchased from Abcam. Anti-GAPDH (1:3000, MAB374) and anti-RAD51C (1:1000, NB100–177) antibodies were purchased from Millipore and Novus, respectively. Anti-Lig3 (1:2000, 611876) and anti-XRCC4 (1:500, sc-8285) antibodies were purchased from BD biosciences and Santa Cruz, respectively. Anti-Artemis (1:3000) and anti-PTIP (1:2000) antibodies were gifts from Dr. Junjie Chen and Dr. Zihua Gong (MD Anderson Cancer Center). Uncropped immunoblots are shown in Supplementary Fig. 8.