Anti ctip

Antibody recognizing 53BP1 (A300-272A) and RPA32 pS (4/8) (IHC-00422) were purchased from Bethyl Laboratories (Montgomery, TX). The phospho-specific γ-H2AX antibody (JBW301) was obtained from Millipore (Billerica, MA). Mre11 (12D7), Ku70 (GTX233114) and Ku80 (GTX70485) were purchased from Genetex. Actin (C-11), BRCA1 (sc-642). RNAPII (sc-899), ATR pS 428 (sc-109912) and Rad51 (H-92) antibodies were obtained from Santa Cruz Biotech (Santa Cruz, CA). Total (2662) and pT68 Chk2 (2661) antibodies and total (2360) and pS317 (2344) Chk1 were purchased from Cell Signaling. S9.6, an antibody specific for R loops (RNA:DNA hybrids) [37 (link)], was provided by Dr. Stephen H. Leppla (NIH, Bethesda, MD). Antibodies used for ChIP: anti-BRCA1 (Gene-Tex, 6B4), anti-ATM (Novus Biologicals NB100-305), anti-γ-H2AX (Abcam, ab2893), anti-53BP1 (Novus Biologicals, NB100-305), anti-CtIP (Abcam) and anti-Gal4(DBD) (Santa Cruz Biotechnology, sc-577). Antibodies used in western blotting and SETX IP: anti-SETX (A301-105) from Bethyl Laboratories, anti-XRN2 (NBP1-68149) and anti-Rrp45 (NBP1-71702) from Novus Biologicals.

Total proteins of thecells were extracted by lysis buffer (Tris-HCl, PH 8.0, 400 mM NaCl, 5 mM EDTA, 1 mM EGTA, 1 mM Na pyrophosphate, 1% Triton X-100, 10% glycerol), with supplement of protease inhibitors (Roche, Indianapolis, IN). The protein concentration was determined by a BCA kit (Piece, Rockford, IL). Then protein was loaded onto 12% SDS-PAGE gel and was electronically transferred to PVDF membranes (Millipore, Billerica, MA). Primary and secondary antibodies were then incubated to the membranes. The HRP-conjugated secondary antibody (1:5000, Sigma-Aldrich) was incubated for 2 h atroom temperature. Protein signals were detected via ECL method. The primary antibodies used in this study includes anti-BRD4 (1:1000, Abcam), anti-c-Myc (1:1000, Abcam), anti-CyclinD1 (1:1000, Abcam), anti-CDK4 (1:1000, Abcam), anti-MMP2 (1:1000, Abcam), anti-MMP9 (1:1000, Abcam), anti-CtIP (1:1000, Abcam), anti-CD274 (1:1000, Abcam), anti-KRAS (1:500, Abcam), anti-E-cadherin (1:10000, Abcam), anti-N-cadherin (1:1000, Abcam), anti-Vimentin (1:3000, Abcam) and anti-GAPDH (1:1000, Abcam). All the primary antibodies were incubated for 4 h at 37 °C.

Human DNA2 and CtIP were detected using anti-CtIP (1/1000, rabbit, Bethyl) and anti-DNA2 (1/1000, rabbit, Abcam). To detect protein interaction in vivo, cells (1 x 10⁷ cells for U2OS and ATLD2) exposed to 6 Gy γ-ray irradiation were incubated for 1 hour. Lysate was prepared from cells pretreated with paraformaldehyde (final concentration 0.05%) for 10 minutes before collection. The lysis buffer contained HEPES-KOH (ph7.5), 300mM KCl, 0.1% Triton X-100, and protease cocktail (Complete, Roche). CtIP and Dna2 were detected using anti-rabbit IgG conjugated with HRP (RPN4301, GE Healthcare).