Anti uhrf1

Whole-cell lysates were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. The resolved proteins were transferred to a polyvinylidene fluoride membrane (0.45 μm pore size). The membrane was probed with primary antibodies: anti-FLAG-M2 (1:2,000; Sigma, St. Louis, MO, USA), anti-MYC (1:500; laboratory-made or 1:2,000; Roche), anti-RTA (1:500; laboratory-made), anti-K8 (1:500; laboratory-made), anti-GFP (1:500; Santa Cruz Biotechnology, Dallas, TX, USA), anti-PARP1 (1:1,000; BD Biosciences), anti-PAR (1:500; Trevigen), anti-CHFR (1:1,000; Cell Signaling Technology, Danvers, MA, USA), anti-UHRF1 (1:500; Santa Cruz Biotechnology), anti-H2AX (1:500; Cell Signaling Technology, Danvers, MA, USA), anti-γH2AX (1:500; Merck Millipore, Billerica, MA, USA) or anti-α-tubulin (1:2,000; Sigma). The membrane was then incubated with the horseradish peroxidase–conjugated goat anti-rabbit or goat anti-mouse immunoglobulin G antibody (1:5000; a secondary antibody; Santa Cruz Biotechnology). The protein bands were detected with enhanced chemiluminescence (ECL) and western blotting detection reagents (ELPIS, Taejeon, Republic of Korea). The protein bands were documented on an LAS-4000 chemiluminescent image analyzer (Fujifilm). The band intensities were calculated in the ImageJ software [72 (link)].

Protein levels were assessed by western blot. Antibodies against HDAC, PARP, cleaved-PARP, H3, Acetyl-H3, and RAD51 were purchased from Cell Signaling Technology (CST). Anti-UHRF1, Ku-70, ERCC1, MSH2, MSH6, GAPDH and anti-α-tublin antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-BRCA1 and phosphorylated-BRCA1(ser988) was purchased from ABclonal Technology (Upper Heyford, UK). Anti-PAR antibody was purchased from Trevigen (4335-MC-100, MD, USA).

The cells were lysed in IP Lysis buffer (87788, Thermo Scientific) along with a protease inhibitor cocktail (Roche Applied Science, Upper Bavaria, Germany) at 4 °C for 30 min. Thereafter, the cell extract was centrifuged at 14,000 rpm for 15 min at 4 °C, and 5% of the cell extract was kept for input. To conjugate the primary antibody, the cell extracts were incubated with 2 µg of the anti-UHRF1 (Santa Cruz), anti-MYC (Cell Signaling), or rabbit IgG antibody and Dynabeads protein G (Thermo Fisher Scientific) for overnight incubation at 4 °C. The antibody–antigen complex was washed three times with PBS. Laemmli buffer (Bio-Rad, Hercules, CA, USA) was subsequently added to elute the precipitated proteins, followed by Western blotting using anti-UHRF1 (sc-373750, Santa Cruz) and anti-c-Myc antibodies (5605S, Cell Signaling). The immunoblot conditions were the same as described for the siRNA-treated samples.