Rad21 antibody

Total proteins were extracted from cells using RIPA buffer (Cell Signaling Technology) and quantified using the BCA Protein Quantification Kit (Abbkine, Wuhan, Hubei, USA). The same amount of protein was loaded and separated by SDS-polyacrylamide gel electrophoresis (SDS‒PAGE) and then transferred to a PVDF membrane. Subsequently, membranes were blocked with 5% nonfat milk for 1 h at RT and then incubated overnight at 4 °C with the primary antibody. The membranes were then washed with Tris-buffered saline containing 0.1% Tween 20 (TBST) and incubated with the corresponding secondary antibodies (1: 5000, BOSTER, Wuhan, Hubei, China) at RT for 1 h. Finally, the signals of the targeted proteins were detected by a chemiluminescence detection kit (Beyotime Biotechnology). The primary antibodies used in this study included NIPBL antibody (#ab245539, Abcam), RAD21 antibody (#4321, Cell Signaling Technology), lysine demethylase 6B (KDM6B) antibody (#ab38113, Abcam), EZH2 antibody (#21800-1-AP, Proteintech), PI3K antibody (#bs-2067R, Bioss, Beijing, China), phospho-PI3K (Tyr317) antibody (#bs-5570R, Bioss) and GAPDH (#KC-5G5, Aksomicks, Shanghai, China).

In brief, H1299 and H1650 cells were crosslinked by 1% formaldehyde for 15 min at RT before the reaction was stopped by adding 1.375 M glycine. Next, the cells were suspended in lysis buffer and sonicated to shear the DNA. The insoluble material was removed by centrifugation. A 25 mg DNA chromatin sample was adjusted to a total volume of 500 mL in 450 mL of dilution buffer containing protease inhibitors. The chromatin samples were then incubated with 1 μg of NIPBL antibody (#ab245539, Abcam), RAD21 antibody (#4321, Cell Signaling Technology, Danvers, MA, USA), H3K27me3 antibody (#21800-1-AP, Proteintech, Rosemont, IL, USA), enhancer of zeste 2; catalytic subunit of polycomb repressive complex 2 (EZH2) antibody (#21800-1-AP, Proteintech) or anti-rabbit IgG antibody (Cell Signaling Technologies) and incubated with protein A/G magnetic beads overnight at 4 °C with gentle rotation. Magnetic beads were collected by a magnetic separation device (Thermo Fisher Scientific) and cleaned using washing buffer. Subsequently, immunoprecipitated DNAs were eluted with 100 μL elution buffer containing Proteinase K at 62 °C for 2 h. The DNAs were then purified using the spin columns and dissolved in the elution buffer. Finally, chromatin DNA was analyzed by PCR or qPCR.