Ab21382

Cells grown on glass coverslips were fixed in 4% paraformaldehyde/PBS for 15 min, then permeabilized with 0.1% triton-X100/PBS at 37°C for 30 min, and finally blocked with 5% goat serum/PBS at 37°C for 3 h. Immunofluorescence was performed using standard methods, and the slides were incubated alternately with BG4 (80 ng/μL), anti-FLAG antibody (#2368, Cell Signaling Technology), γH2AX antibody (#9718, Cell Signaling Technology), TRF2 antibody (ab13579, Abcam), or POT1 antibody (ab21382, Abcam) at 37°C for 3 h. The glass coverslips were washed six times with blocking buffer and were then incubated with anti-rabbit Alexa 488-conjugated antibody (A21206, Life Technology), anti-mouse Alexa 555-conjugated antibody (A21427, Life Technology), and 2 μg/mL of 4′,6-diamidino-2-phenylindole (DAPI, Invitrogen) at 37°C for 3 h. The glass coverslips were again washed six times with blocking buffer, and then, digital images were recorded using an LSM710 microscope (Zeiss, GER) and analyzed with ZEN software. Fifty nuclei were counted in each group, and the s.e.m. was calculated from three replicates. Frequency distribution graphs were plotted using GraphPad Prism (GraphPadSoftware Inc.).

The ChIP assays were performed according to the manufacturer’s protocol (Upstate Biotechnology, Inc., Lake Placid, NY), with the exception of the conditions for sonication that were changed to five times for 30 seconds each at 10% output. The antibodies used for the immunoprecipitations were anti-TRF2 (ab13579, abcam), anti-TRF1 (ab1423, abcam), anti-POT1 (ab21382, abcam), anti-HA (ab9110, abcam), anti-hist1H2AC (Novus Biologicals), anti-Histone H3.3 (ab62642, abcam) anti-γ-H2AX (pS-139) (ab2893, abcam), anti-ATM (pS-1981) (ab36810, abcam) and anti-XPF (ab17798, abcam). Non-specific rabbit polyclonal antibodies were used as a negative control. After reversing the protein-DNA cross-linking of the immunoprecipitated complexes, DNA was extracted for dot blotting analysis.