Anti ku70

For native RIP, MDA-MB-231 extract was incubated with 10 μg of anti-Ku70 (Cat No:MA5-13110, Clone No:N3H10, Thermo), anti-Ku80 (Cat No:MA5-12933, Clone No:111, Thermo), anti-DNA-PKcs (Cat No: MA5-13404, Thermo) antibody or control IgG (Cat No:5415S, CST) and then with Protein A Sepharose beads. After a total of three washes in RIP buffer, beads were boiled in SDS buffer for Western blot, or resuspended in TRIzol reagent for real-time RT-PCR. UV-Crosslink RIP (CLIP) was performed as described^{34 (link)–36 (link)}. Briefly, UV-irradiated MDA-MB-231 cells were lysed in RSB-Triton buffer, incubated with anti-Ku70 (Cat No:MA5-13110, Clone No:N3H10, Thermo), anti-Ku80 (Cat No:MA5-12933, Clone No:111, Thermo), anti-DNA-PKcs (Cat No: MA5-13404, Thermo) antibody or control IgG (Cat No:5415S, CST), and then precipitated with Protein A Sepharose beads. Beads were then extracted for Western blot or real-time RT-PCR.

Cells were lysed in mammalian protein extraction reagent (Pierce). After quantification using a BCA protein assay kit (Pierce), total proteins were separated by SDS-PAGE under denaturing conditions and transferred to PVDF membranes (Millipore). Membranes were blocked in 5% non-fat milk (Bio-Rad) and then incubated with anti-CDK7 (Cat No: sc-7344, Santa Cruz Biotechnoloy); anti-BRCA1 (Cat No: sc-6954, Santa Cruz); anti-BRCA2 (Cat No: OP95, Millipore); anti-RAD51 (Cat No: ab213, Abcam); anti-Ku80 (Cat No: MA5–12933, Thermo); anti-Ku70 (Cat No: MA5–15110, Thermo); anti-RNAP II (Cat No: sc-17798, Santa Cruz Biotechnoloy); anti-RNAP II p-Ser5 (Cat No: A300–655A-2, Bethyl); or anti-phospho-H2AX(S139) (Cat No:05–636, Clone No: JBW301, Millipore), followed by incubation with secondary antibodies conjugated with horseradish peroxidase (HRP, GE Healthcare Life Sciences). Anti-b-Tubulin (Cat No: 2128, Clone No: 9F3, CST) or anti-Actin (Cat No: A3854, Sigma) was used for internal loading control. Immunoreactive proteins were visualized using the LumiGLO chemiluminescent substrate (Cell Signaling).