Patr s428

Cells were harvested by scraping or trypsinization and lysed with buffer [50 mM Tris–HCl, pH 7.8, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1X protease/phosphatase inhibitor cocktail (Thermo 1861280)]. 2X SDS loading buffer was added to the lysates before heating at 95°C for 5 min.
Three to eight percentage Tris-Acetate SDS PAGE gradient gels (Invitrogen EA0378) were used to resolve ATR-H from ATR-L; otherwise, standard SDS PAGE gels were used. PVDF membranes (Amersham 10061-492) were used to capture proteins transferred from gels. Chemiluminescent signal was captured using the GE Amersham Imager 680. Antibodies and their dilutions for western blots include pATR S428 (Cell Signaling 2853) 1:1000, PP2Ac (Cell Signaling 2038) 1:1000, GAPDH (Cell Signaling 5174) 1:1000, PARP 1 (Cell Signaling 9532) 1:1000, Bid (Cell Signaling 2002) 1:1000, or (Santa Cruz Biotechnology sc6538) 1: 500, Beta Actin (Invitrogen MA1-140) 1:5000, mtHSP70 (Invitrogen MA3-028) 1:1000, ATR (Bethyl Laboratories, Inc., A300-137A, A300-138A) 1:8000, ATRIP (ABclonal A7139) 1:1000, PP2A-Cα/β (Santa Cruz Biotechnology sc-80665) 1:500, Cleaved Caspase-3 (Cell Signaling 9664) 1:1000, PP4c (Bethyl Laboratories, Inc., A300-835A) 1:8000, PP5c (Bethyl Laboratories, Inc., A300-909A) 1:8000.

Cells or tissue specimens were lysed in UREA buffer (8 M Urea, 1 M Thiourea, 0.5% CHAPS, 50 mM DTT, and 24 mM Spermine). Same amount of proteins were separated by SDS-PAGE. After being incubated with the indicated antibodies, the immune complex on membrane was detected by an ECL kit (Thermo Scientific, #32106). Antibodies used for WB were shown as follows: Antibodies against p-ATM (S1981) (#Ab81292), ATM (#Ab32420), NBS1 (#Ab32074), and RAD50 (#Ab89) were from Abcam. Antibodies against p-Chk2 (T68) (#2197), Mre11 (#4847), ATR (#13934), and p-ATR (S428) (#2853) were from Cell Signaling Technology. Antibodies against Chk2 (#13954-1-AP), FLAG (#20543-1-AP, for WB), RAD51 (#14961-1-AP), XRCC4 (#15817-1-AP), α-tubulin (#66031-I-Ig), β-actin (#60008-1-Ig), and GAPDH (#60004-1-Ig) were obtained from Proteintech. Additional antibodies included EGR1 (sc-189, Santa Cruz), Histone3.1 (#KM9005T, Sungene), FLAG (#MCA4764, Bio-Rad, for IP), mouse IgG (#A7028, Beyotime), and Rabbit IgG (#A7016, Beyotime).

Whole cell extracts were prepared by lysing cells in two volumes of Lysis Buffer (250 mM NaCl, 5 mM ethylenediaminetetraacetic acid (EDTA), 50 mM HEPES, 0.1% v/v NP40). A total of 60 μg of whole cell extract (WCE) was resolved on 4–12% NOVEX gels using MOPS buffer (Invitrogen). Protein was transferred to PVDF membrane (Invitrogen) and blotted for BCLAF1 (BTF 608A, Bethyl Labs); THRAP3 (A300–956A, Bethyl Labs), GAPDH (ab9485, Abcam), pATM (S1981) (ab79891 Abcam), ATM (2C1, Santa Cruz), pATR (S428) (2853, Cell Signaling), ATR (N-19, Santa Cruz), 53BP1 (NB100–34, Novus Biologicals), γH2AX (S139) (JBW301, Millipore), cyclin B1 (GNS1) (sc-245, Santa Cruz), pP53 (S15) (9284S, Cell Signaling), pKAP1 (S824) (A300–767A, Bethyl Labs), FANCD2 (FI17) (sc200–22, Santa Cruz), BRCA2 (H-300) (sc-8326, Santa Cruz), FANCL (B-11) (sc-137067, Santa Cruz), PALB2 (H-45) (sc-382436, Santa Cruz) and Rad51 (3C10) (sc-53428, Santa Cruz).