P dna pkcs

Post-irradiation, at 0 h, 0.5 h, and 8 h, preparation of total cell lysates was performed using the ProtectJETTM Mammalian Cell Lysis Reagent from Fermentas (Lithuania) as per the protocol provided by the manufacturer. The membranes with the transferred protein were incubated with gentle agitation with following specific primary antibodies (1 : 1,000) at 4°C overnight: p-ATM (1 : 1000), p-DNA-PKcs (1 : 1000), Rad51 (1 : 1000), MMP10 (1 : 1000), and actin (1 : 1000) (all primary antibodies were from Abcam, USA). The secondary antibody (1 : 5000) were also from Abcam. Electrochemiluminescence (Santa Cruz Biotechnology Inc) was used to detect all the membranes.

After the cells were treated with different concentrations of HS-173 and irradiated for various times, they were collected and washed with cold phosphate-buffered saline (PBS). The cells were then lysed with RIPA buffer containing protease and phosphatase inhibitor cocktails (GenDEPOT, Barker, TX, USA). Proteins in whole-cell lysates were resolved by sodium dodecyl sulfate protease and phosphatase inhibitor (SDS-PAGE), and transferred onto nitrocellulose membranes. The blots were first incubated with the appropriate primary antibodies, and then with horseradish peroxidase (HRP)-conjugated secondary antibodies. Antibody binding was detected using enhanced chemiluminescence reagents (Bio-Rad, Hercules, CA, USA). Primary monoclonal antibodies against the following proteins were used: cleaved PARP, cleaved caspase-3, survivin, p-AKT, AKT (Cell Signaling Technology, MA, USA) p-Cdc2, p-ATM, ATM, p-DNA-PKcs, DNA-PKcs, p-KAP1, p-53BP1 (Abcam, Cambridge, UK) KAP1, 53BP1(Santa Cruz, Dallas, TX, USA) and β-actin (Sigma-Aldrich, OH, USA) Secondary antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Total cellular proteins were isolated following a 48 hour-treatment with 1.0 μM of MST-312 and/or 10 μM of NU7026, using RIPA (radio-immunoprecipitation assay) buffer (1% nonidet P-40, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate, 2 mM EDTA, 50 mM sodium fluoride, 0.2 mM sodium vanadate and 100 U/ml aprotinin, pH 7.2) from control and treated cells. The whole cell lysate was recovered by centrifugation at 14,000 rpm for 10 minutes. Protein concentration was determined by the bicinchoninic acid method using an assay kit (Pierce Biotechnology, USA) with bovine serum albumin as a standard. Western blot analyses of, DNA-PKcs, p-DNA-PKcs(Ser2056), (phospho DNA-PKcs, Abcam, UK,1:1000) hTERT (Epitomics, USA), and β-actin were performed using specific antibodies from Santa Cruz Biotechnology, USA unless otherwise stated.