Brca1 sc 6954

Cell lysates were generated by lysis in NETN buffer (50 mM Tris-HCl pH = 7.4, 250 mM NaCl, 5 mM EDTA, 0.1% NP-40, 10 mM NaF, 200 μM Na₂VO₃, 0.5 mM PMSF, and 1x protease inhibitor cocktail) on ice for 30 min. Non-soluble debris was precipitated by a 5 min 12000 rpm spin at 4 °C and discarded. Lysate was treated with 4x loading buffer (0.5 M Tris-HCl pH 6.8, 277 mM SDS, 40% glycerol, bromophenol blue, 4% 2-mercaptoethanol), ran on a 10% SDS-PAGE gel, and transferred onto a PVDF membrane for 1–2 hr at 500 mA. The following Santa Cruz (Dallas, TX, USA) antibodies were used: BRCA1 (sc-6954) and RAD51 (sc-8349 and in-house aliquot B32). The following Cell Signaling (Danvers, MA, USA) antibodies were used: ATM (2873), GAPDH (5174), Chk2 (2662), pChk2 T68 (2661), Chk1 (2345), and pChk1 S345 (2341). Other antibodies included DEK (610948 BD Bioscience, San Jose, CA, USA), DEK (16448-1-AP, Protein Tech, Rosemont, IL, USA), DEK (in-house K-877)72 (link), DNA-PKcs (ab1832, Abcam, Cambridge, MA, USA), pDNA-PKcs S2056 (ab18192), pATM S1981 (AF1655, R&D Systems, Minneapolis, MN), γH2AX (05-636, Millipore, Darmstadt, Germany), MRE11 (GTX70212, genetex, San Antonio, TX, USA), and NBS1 (NB100-143SS, Novus Biologicals, Littleton, CO, USA).

Olaparib (AZD2281, Ku-0059436) was obtained from Targetmol (Shanghai, China). Proguanil was obtained from Selleck Chemicals (Shanghai, China). Acridine orange/ethidium bromide (AO/EB) staining was obtained from Kehang Biotechnology (Changsha, China), and the FITC Annexin V Apoptosis Detection kit was from BD Pharmingen (NJ, USA). Rabbit anti-human antibodies specific for the following proteins were obtained from Cell Signaling (MA, USA): Phospho-Histone H2A.X (Ser139) and β-actin. Phospho-ATM (Ser1987) was obtained from Abcam (MA, USA). BRCA1 (sc-6954) was obtained from Santa Cruz Biotechnology (Texas, USA). Bcl-2 polyclonal antibody and Bax polyclonal antibody were obtained from Bioworld Technology (MN, USA) and Biyotime (Shanghai, China) respectively.

To analyze protein expression in cells, immunoblotting analysis was performed as previously described [2 (link)]. Antibodies against the following proteins were obtained from Santa Cruz Technology (California, US): Aur A (sc-25425), Aur B (sc-25426), BRCA1 (sc-6954), CDK2 (sc-163), CDK4 (sc-260), CDK6 (sc-177), cyclin D1 (sc-718), cyclin E (sc-247), cyclin A (SC-751). Antibodies against BRCA2 (19791-1-AP) and cyclin B1 (cs-4135) were obtained from Proteintech (Chicago, USA) and Cell Signaling Technology (Massachusetts, US), respectively. The detection of β-actin (A2228, Sigma Aldrich, St. Louis, MO) was used as a loading control. The secondary antibodies were F(ab)2 fragments of donkey anti-mouse immunoglobulin or of donkey anti-rabbit immunoglobulin linked to horseradish peroxidase from Cell Signaling Technology (Massachusetts, US). Immunoblotting reagents were from an electrochemiluminescence kit (Amersham Biosciences). To test whether the expression levels of proteins were regulated through proteasome-mediated degradation, cells were exposed with 20 μM MG132 (#S2619, Selleck Company, Texas, America) for 3 h and then harvested for Western blotting analysis.