Patm s1981

Standard protocol was followed for Western blot analysis. Cell lysates were collected in 200 μl of Laemmli buffer, vortexed, and boiled at 95°C for 5 min. Protein concentrations were measured by reducing agent-compatible BCA assay. SDS-PAGE was followed by transfer onto a PVDF membrane, which was then blocked in 5% BSA. Primary antibodies against the following proteins were used: nucleolin (mouse monoclonal; Santa Cruz Biotechnology, Santa Cruz, Calif., USA), ATM and pATM S1981 (rabbit polyclonal and mouse monoclonal, respectively; Rockland, Gilbertsville, Pa., USA), Chk2 and pChk2 T68 (rabbit polyclonal and mouse monoclonal, respectively; Cell Signaling). Blots were stained with secondary antibodies and visualized with the Odyssey near-infrared system (LI-COR, Lincoln, Nebr., USA).

The following antibodies were used: Phospho-ATM/ATR Substrate Motif (pS/TQ) (1:1,000; Cell Signaling, #6966), NIPBL (1:1,000; Santa Cruz, sc-374625), WAPL (1:1,000; Cell Signaling, 77428S), PDS5A (1:1,000; Bethyl A300–089A), PDS5B/APRIN (1:1,000; Novus, NB100–755), Flag (1:1,000; Sigma, F1804), SMC1A (1:1,000, Bethyl A300–055A), pSMC1A S957 (1:1,000 [WB], 1:200 [IF]; Cell Signaling, 4805S), 53BP1 (1:200 [IF], Bethyl, A300–237A); γH2AX (1:200 (IF); Millipore 05–636), ATM (1:1,000; Sigma, A1106), pATM S1981 (1:1,000, Rockland, 200–301–400), CHK2 (1:1,000, Cell Signaling, 2662), pCHK2 T68 (1:1,000; Cell Signaling, 2661), DNAPK (1:1,000; Abcam, ab70250), pDNAPK S2056 (1:1,000; Abcam, ab18192), CHK1 (1:1,000, Epitomics, 2865–1), pCHK1 S345 (1:1,000, Epitomics, S0660). For immunoblotting proteins were transferred to nitrocellulose after separation on an acrylamide gel, blocked in 5% milk in TBS/tween. Primary antibodies were diluted at the indicated dilutions above in 5% bovine serum albumin (BSA) in TBS/t. Proteins were visualized by staining with Alexfluor 700 or 800 conjugated secondary antibodies on an Odyssey CLx imaging system.