Anti phospho nbs1 s343

The following antibodies were used in this study: anti-FLAG (F3165, Sigma, St Louis, MO, USA); anti-BRCA1 (D-9, Santa Cruz, Dallas, TX, USA); anti-cyclin A (sc-751 and sc-271645, Santa Cruz); anti-NBS1 (A301-289A, Bethyl, Montgomery, TX, USA); anti-53BP1(612522, BD, San Jose, CA, USA); anti-phospho-ATM (S1981) (4526, Cell Signaling, Danvers, MA, USA); anti-phospho-RPA32 (S4/S8) (A300-245, Bethyl); anti-phospho-53BP1 (S25/29) (2674, Cell Signaling); anti-phospho-53BP1 (S1778) (2675, Cell Signaling); anti-phospho-NBS1 (S343) (3001, Cell Signaling); anti-phospho-SMC1 (S957) (4801, Cell Signaling) anti-phospho-CHK1 (S345) (2348, Cell Signaling); anti-phospho-KAP-1 (S824) (4127, Cell Signaling) and anti-phospho-(Ser/Thr) ATM/ATR substrate antibody (2851, Cell Signaling). Rabbit anti-PTIP antisera were obtained from rabbits immunized with GST-PTIP (residues 590–1 069) fusion protein expressed and purified from Escherichia coli. Antisera were affinity-purified using AminoLink Plus Immobilization and Purification Kit (Pierce, Waltham, MA, USA ). Rabbit polyclonal antibodies against BRCA1, RIF1, ATM, γH2AX, 53BP1 and phospho-CHK2 (T68) were described previously [5 (link), 31–33 ].

Whole-cell extracts were prepared in lysis buffer and protein concentration was determined using the BCA Protein Assay Reagent Kit (Pierce, Rockford, IL, USA). Fifty micrograms of total proteins were loaded onto 10–13% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred onto nitrocellulose membranes. Membranes were incubated with primary antibodies overnight at 4 °C with gentle rocking followed by horseradish peroxidase-conjugated secondary antibody for 1 h. Chemiluminescence signals were visualized using Western blotting luminol reagent (Santa Cruz Biotech, Santa Cruz, CA, USA) and exposed to film. The blots were screened/quantified with the software Quantity One (Bio Rad, Hercules, CA, USA) and normalized against β-Actin level. The target protein/Actin value obtained from control (8-Cl-Ado-exposed for 0 h) cells was designated as “1”. Anti-p21, anti-p53, anti-p53R2, anti-phospho-p53-S15, anti-CHK1-S345, anti-CHK2-T68, anti-CHK1, anti-CHK2, anti-ATR, anti-phospho-ATR-S428, anti-NBS1, anti-phospho-NBS1-S343, anti-SMC1, anti-phospho-SMC1-S699, anti-β-actin, anti-BRCA1 and anti-phospho-BRCA1-S1524 antibodies were purchased from Cell Signaling Technology; anti-phospho-histone H2AX-S139, anti-ATM and antiphospho-ATM-S1981 were acquired from R&D Systems Inc. (Minneapolis, MN, USA).