Anti chfr

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)].

Paclitaxel was purchased from Bristol-Myers Squibb Pharmaceutical Ltd. MG-132(#S2619) was purchased from Sigma. The antibodies used for Western blotting included anti-UbC13 (#ab25885, Abcam), anti-DNMT1 (#24206-1-AP, Proteintech), anti-CHFR (#6904, Cell Signaling Technology), anti-Aurora A (#12100, Cell Signaling Technology), anti-Ub (#3933, Cell Signaling Technology), anti-HA(#ab003, Lianke), anti-EGFP (#ab006, Lianke), anti-GAPDH (#Mab5465, Lianke), anti-mouse HRP (#7076, Cell Signaling Technology), and anti-rabbit HRP (#7074, Cell Signaling Technology). Antibody anti-DNMT1 (#ab13537, Abcam) was used for immunoprecipitation.

HeLa cells were seeded onto a cover glass in a 24-well plate for 24 h before transfection. The DNA constructs were transfected into the cells by the PEI transfection method for 48 h. The cells were fixed for 15 min with 4% paraformaldehyde and 0.15% picric acid in phosphate-buffered saline (PBS) and blocked with 10% normal goat serum prepared in PBS containing 0.3% of Triton X-100 and 0.1% of bovine serum albumin. The TPA or DMSO treated BC-3 cells were harvested, washed with PBS, and fixed for 10 min in cold acetone. The fixed cells were washed again with PBS and air-dried. Then the cells were blocked with 10% normal goat serum prepared in PBS containing 0.3% of Triton X-100 and 0.1% of bovine serum albumin. Next, the cells were incubated with the anti-MYC (1:200), anti-PF-8 (1:100; a kind gift from Dr. Bala Chandran at University of South Florida (Tampa, Florida, USA)), anti-CHFR (1:100), and anti-PARP1 (1:800; Cell Signaling Technology) antibodies for 16 h at 4°C, followed by probing with secondary antibodies (anti-mouse-Cy3 and anti-rabbit-Rho; 1:2,000; Jackson Immuno Research, West Grove, PA, USA) for 45 min at room temperature. After that, the cells were incubated with 4′,6-diamino-2-phenylindole (DAPI; 1:1000) for nuclear staining. Fluorescence images were captured at a magnification of 1000× under a confocal laser scanning microscope (LSM 5 Exciter, Zeiss).