Phospho chk2 2197

The antibody against BHRF1 was a gift from Jaap Middeldorp (VU University Medical Centre, The Netherlands). Antibodies against BZLF1 and BRLF1 were purchased from Argene (bioMérieux SA, Marcy l'Etoile, France). The rabbit monoclonal antibodies against phospho‐ATM (ab81292), ATM (ab32420), and the anti‐HA tag (1:5000 dilution; ab9110) rabbit polyclonal antibody were purchased from Abcam. The anti‐γ‐H2AX^ser139 antibody was purchased from EMD Millipore (Quincy, MA, USA). The antibodies against CHK2 (#3440) and phospho‐CHK2 (#2197) were purchased from Cell Signaling Technology (Danvers, MA, USA). All of the AlexaFluor‐conjugated and HRP‐conjugated secondary antibodies were purchased from Molecular Probes (New York, NY, USA). The HRP‐conjugated secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Western blot analysis was performed as previously described 24 and all the primary and the secondary antibodies used is 1:1000 and 1:5000 in dilutions unless otherwise specified. The signal intensity was measured by ImageJ software (http://rsb.info.nih.gov/ij).

The cells were plated and treated as described above for 48 hours, and then were lysed in cell lysis buffer (Beyotime, P0013, Beijing, China) supplemented with 0.5 mM phenylmethanesulfonyl fluoride (PMSF, Beyotime, ST506). The total cellular protein concentration was determined with a BCA Protein Assay Kit (Thermo Fisher Scientific Inc., 23227, Rockford, USA). The proteins were applied to sodium dodecyl polyacrylamide gel electrophoresis, and transferred onto a PVDF membrane (Millipore, Billerica, MA, USA). Then membranes were blocked with 5% evaporated skimmed milk for 1 hour and probed overnight at 4°C with the following primary antibodies: cleaved-CASP 3 (9664), Cytochrome C (4280), Phospho-Chk1 (2348), Phospho-Chk2 (2197), Phospho-ATM (5883), Phospho-Histone H2A.X (9718), BAX (2772), Bcl-2 (2870), (all 1:1000; Cell Signaling Technology, Danvers, MA, USA), antibody against ACTB (1:2000; Zsbio, sc-53142, Beijing, China), followed by incubation with horseradish peroxidase coupled secondary anti-mouse (Zsbio, ZB-2305) or anti-rabbit antibodies (Zsbio, ZB-2301) for 1 hour at room temperature. The protein bands were visualized using ECL blotting detection reagents (Beyotime, P0018), and developed and fixed onto x ray films. ACTB was served as a loading control.