P nbs1 s343

Protocol to separate cytosolic and nuclear for immunoblotting was as previously reported [47 (link)]. In brief, B cells (1 × 10⁷) were lysed in 20 µl buffer A (10 mM Hepes, 10 mM KCl, 1.5 mM MgCl₂, 0.34 M sucrose, 10% glycerol, 1 mM DTT, 0.1% Triton X-100, with protease inhibitor freshly added) and left on ice for 5 min. The lysate was centrifuged by 1300 × g, 4 °C for 4 min to receive nuclei pellet. The supernatant was further clarified by 20,000 × g, 4 °C for 15 min to remove cell debris and insoluble aggregates and receive the cytosolic fraction. Nuclei were washed once in buffer A, and then lysed in 30 µl buffer B (3 mM EDTA, 0.2 mM EGTA, 1 mM DTT, and freshly added protease inhibitors). After centrifugation by 1700 × g, 4 °C for 5 min, the yielded supernatant as nuclear fraction was collected. In-house anti-PP4 antibody was generated as described previously [42 (link)]. Antibodies recognizing p-p53 S15 (#9284), p-ATR S428/S431 (#2853), p-Chk1 S345 (#2348), p-NBS1 S343 (#3001), NBS1 (#14956), γH2AX (#9718), or H2AX (#7631) were from Cell Signaling. Antibodies recognizing ATM (2C1), p-ATM S1981/1987 (10H11.E12), GAPDH (GTX100118), or p84 (5E10) were from GeneTex; and anti-p53 (SC-6243) was from Santa Cruz. ECL Plus^TM Western Blotting Detection Reagents (Amersham) were utilized for immunoblot development.

Western blotting was performed as previously described [64 (link)]. Equal amounts of cell lysates or immunoprecipitates were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subjected to western blotting using the following antibodies. Primary antibodies against P-gp, Csk and FANCD2 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA), Src, pSrc^Y416, pSrc^Y527, ATM, pATM^S1981, Chk2, pChk2^T68, Brca1, pBrac1^S1524, Nbs1, pNbs1^S343, Mre11 and Rad50 from Cell Signaling Technology (Danvers, MA, USA), Rad51 from GeneTex (Irvine, CA, USA), γH2AX from Calbiochem (San Diego, CA, USA), and Cbp and β-actin from Abcam (Cambridge, MA, USA). The secondary horseradish peroxidase-conjugated antibody was purchased from Abcam (Cambridge, MA, USA). The specific protein bands were visualized by chemiluminescence using the SuperSignal West Pico chemiluminescence reagent (Pierce, Rockford, IL).