Anti lig4

Whole-cell extracts were collected in SDS sample buffer and boiled for 10 mins. Samples were resolved by SDS-PAGE, transferred to PVDF, and blocked with 5% milk in PBST (PBS, 0.1% Tween-20). The following primary antibodies were used at 1:1,000 dilution (unless noted) in PBST: anti-CENP-A (Cell Signaling, 2186), anti-phospho histone H2AX (ser139) clone JBW301 (EMD Millipore, 05-636), anti-phospho histone H3 (ser10) (Cell Signaling, 9706), 1:4,000 anti-histone H3 (Sigma H0164), anti-LIG4 (GeneTex, GTX100100), anti-DNA-PKcs (Bethyl, A300-516A), anti-LIG3 (Bethyl, A301-637A), anti-PARP (BD Pharmingen, 556362, kindly provided by X. Wu, The Scripps Research Institute, USA), anti-BRCA2 (Bethyl, A303-434A), anti-RAD51 (Abgent, AM8421b), and 1:2,000 anti-GAPDH (Cell Signaling, 14C10). Blots were probed with 1:4,000 dilutions of HRP-conjugated secondary antibodies (GE Healthcare) and exposed to film. All unprocessed film scans with the appropriate size markers are provided in Supplementary Fig. 6.

Western blot analysis was performed as previously described(14 (link)). c-MYC and Ku70 (Santa Cruz Biotechnology, Dallas, TX) antibodies were used at 1:500 and 1:1000, respectively. Anti-LIG3 (clone 7, BD Biosciences, San Jose, CA) and anti-PARP1 (Cell Signaling Technologies, Danvers, MA) were used at 1:3000. Anti-LIG4 (GeneTex) was used at 1:1000. Anti-GRB2 (Cell Signaling Technology) was used at 1:1000. Anti-actin antibody (Sigma, St Louis, MO) was used as a loading control at 1:10,000. Secondary antibodies conjugated to HRP (KPL, Gaitherburg, MD) were added at a 1:10,000 dilution. Signal was detected using ECL (GE Healthcare, Pittsburgh, PA). Bands were quantified with ImageQuant software (Bio-Rad, Hercules, CA).

Following antibodies were used in this study at the indicated concentration. Anti-phospho histone H2AX (Ser139) (γH2AX) (clone JBW301, EMD Millipore, 05–636, 1:1500 IF) (Cell Signaling Technologies, 2577, 1:1000 IF), anti-TOPBP1 (Bethyl Laboratories, A300–111A, 1:2000 IF, 1:2000 IB) (Abcam, ab2402, 1:1000 IF), anti-MDC1 (Abcam ab11171, 1:1000 IF, 1:1000 IB), anti-CIP2A (clone 2G10–3B5; Santa Cruz sc80659, 1:500 IF, 1:1000 IB), anti-GAPDH (clone 14C10, Cell Signaling Technologies, 1:5000 IB), anti-PolD3 (clone 3E2, Abnova, H00010714-M01, 1:1000 IB), anti-Lig4 (clone N2C2, GeneTex, GTX100100, 1:1000 IB), anti-Mre11 (clone 12D7, GeneTex, GTX70212, 1:1000), anti-Rad50 (Cell Signaling Technologies, 3427, 1:1000 IB), anti-HA-tag (Novus biologicals, NB600–363, 1:2000 IB), anti-Lamin A/C (clone E-1, Santa Cruz, sc-376248, 1:200 IF), anti-Lamin B1 (clone C-5, Santa Cruz, sc-365962, 1:200 IF), anti-53BP1 (Thermo Fisher Scientific, PA1–16565, 1:1000 IF), anti-BRCA1 (clone D-9, Santa Cruz, sc-6954, 1:1000 IF).