Anti rad21 antibody

H1299 and H1650 cells were lysed in nondenaturing lysis buffer. Next, the supernatant of the cell lysate was precleaned by protein A/G magnetic beads (Thermo Fisher Scientific) for 2 h at 4 °C. Subsequently, approximately 300 μg of protein sample was incubated overnight at 4 °C with 1 μg of anti-NIPBL antibody (#ab245539, Abcam) or anti-RAD21 antibody (#4321, Cell Signaling Technology) and 25 μL of protein A/G magnetic beads. The next day, the protein A/G magnetic beads were collected using a magnetic separation device (Thermo Fisher Scientific), and the precipitated complexes were cleansed with washing buffer (Thermo Fisher Scientific). Finally, the bound proteins were analyzed by Western blotting using KDM6B antibody (#ab38113, Abcam) or EZH2 antibody (#21800-1-AP, Proteintech). Rabbit IgG was used as the negative control.
For competitive Co-IP, different dosages of NIPBL expression vector (0.5 μg, 1 μg, and 2 μg) was transfected into control H1299 or H1650 cells and then the interaction RAD21 and NIPBL or EZH2 was evaluated by Co-IP using anti-RAD21 antibody (#4321, Cell Signaling Technology) as above described.