Escherichia coli BL21(DE3) cells were transformed with pET-32a(+)-zbRIG-I or pET-32a(+) plasmids, respectively. Then, cells were grown in 50 ml of LB medium (Beyotime) containing 0.5 mM isopropyl-1-thio-β-D-galactopyranoside (IPTG) (Sigma) at 18°C overnight with shaking at 120 rpm. Cells were pelleted by centrifugation at 4,500 rpm for 30 min and lysed in 10 ml of lysis buffer (100 mM sodium phosphate, pH 8.0, 600 mM NaCl, and 0.02% Tween-20) (Beyotime) via sonication on ice. The sonicated mixture was centrifuged at 15,000 rpm at 4°C for 20 min, and then the supernatant was affinity-purified with Dynabead His-Tag magnetic beads (Invitrogen) according to the manufacturer's instruction.
His pull-down assays were performed as described previously with some modifications (20 (link)). His-zbRIG-I-magnetic beads were incubated with the lysates of HEK 293T cells transfected with pCMV-Flag-zbTRIM25 or pCMV-Flag empty vectors on a roller, respectively. After incubation at 4°C overnight, the magnetic beads were washed three times with lysis buffer to remove unbound His-zbRIG-I and then analyzed via Western blotting using anti-Flag or anti-His antibodies. His tag protein alone was served as a negative control.
His pull-down assays were performed as described previously with some modifications (20 (link)). His-zbRIG-I-magnetic beads were incubated with the lysates of HEK 293T cells transfected with pCMV-Flag-zbTRIM25 or pCMV-Flag empty vectors on a roller, respectively. After incubation at 4°C overnight, the magnetic beads were washed three times with lysis buffer to remove unbound His-zbRIG-I and then analyzed via Western blotting using anti-Flag or anti-His antibodies. His tag protein alone was served as a negative control.