Enhanced chemifluorescence detection system

Total protein extracts from cells were prepared using RIPA buffer (Beyotime Institute of Biotechnology, Nantong, China). Proteins were fractionated in SDS–polyacrylamide gels, transferred to polyvinylidene fluoride (Millipore, Billerica, MA, USA), and western blotting were performed by using the appropriate antibody. Antibody/antigen complexes were detected using ECL (Western Bright Sirius; Advansta, Inc., Menlo Park, CA, USA) and images were acquired using an enhanced chemifluorescence detection system (Amersham Biosciences, Piscataway, NJ, USA) under the room temperature.
For coimmunoprecipitation, Total protein lysates (0.5 mg) were incubated with 4 μg of specific anti-NFBD1 antibody overnight at 4 °C. NFBD1 immunocomplexes were captured with SureBeadsTM Magnetic Beads (Bio-Rad, Hercules, CA, USA) for 1 h at 4 °C. The resulting immunocomplexes were collected by centrifugation, were washed, boiled in SDS sample buffer, loaded on an SDS–polyacrylamide gel. Proteins were analysed by western blotting using standard methods and detected as described above.

The antibodies used in this study were: mouse and rabbit anti-NFBD1 (Abcam, London, UK); anti-ATM (phospho S1981) (Abcam); anti-DNA PKcs (phospho T2609) (Abcam); anti-phospho-H2AX (Ser139) (Cell Signaling Technology, Danvers, MA, USA); anti-phospho-histone H3 (Ser10) (Cell Signalling Technology); rabbit anti-Rad51 (Santa Cruz Biotechnology, Dallas, TX, USA). Total protein extracts from cells were prepared using RIPA buffer (Beyotime Institute of Biotechnology, Nantong, China). Protein concentrations were determined using the BCA protein assay systems (Beyotime Institute of Biotechnology, Nantong, China). Proteins were fractionated in SDS–polyacrylamide gels. Proteins were transferred to polyvinylidene fluoride (Millipore, Billerica, MA, USA), and western blotting were performed by using the appropriate antibody. Antibody/antigen complexes were detected using ECL (Western Bright Sirius; Advansta, Inc., Menlo Park, CA, USA) and images were acquired using an enhanced chemifluorescence detection system (Amersham Biosciences, Piscataway, NJ, USA) under the room temperature.

Total protein extracts from cells were prepared using RIPA buffer (Beyotime Institute of Biotechnology, Nantong, China), fractionated in SDS-polyacrylamide gels, transferred to polyvinylidene fluoride (Millipore, Billerica, MA, USA), and western blotting were performed by using the appropriate antibody. Antibody/antigen complexes were detected using ECL (Western Bright Sirius; Advansta, Inc., Menlo Park, CA, USA) and images were acquired using an enhanced chemifluorescence detection system (Amersham Biosciences, Piscataway, NJ, USA) under the room temperature.