Sa008100

pGEX-NOP53-mEGFP plasmid and other NOP53 fragments plasmids were transformed into E. coli strain BL21 (DE3) cells, respectively. Cultures were grown at 37 °C until the OD600 reached 0.6–0.8 and then induced by adding 0.5 mM isopropyl beta‐d‐thiogalactopyranoside (IPTG) for growth at 16 °C overnight. The next day, cells were pelleted by centrifugation at 4000 × g for 10 min at 4 °C followed by resuspending in lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10% glycerol and 1 mM dithiothreitol (DTT)) and adding 1 mM phenylmethanesulfonyl fluoride (PMSF) before cells lysing by sonication (power setting of 50%, 120 × 5 s with 5 s intervals). After centrifugation at 10,000 × g, 4 °C for 10 min, the supernatant was incubated with GST-tagged purification resin (SA008100, Smart-Lifesciences, Changzhou, China) at 4 °C for 2 h. Then, resin was washed well with GST lysis buffer and NOP53 protein was eluted with glutathione (GSH) elution buffer [20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10% glycerol, 1 mM DTT and 25 mM GSH]. The eluted protein was digested with human rhinovirus type 14 3 C protease (P2303, Beyotime) and purified by HiTrap Heparin HP/Capto HiRes Q/Superdex 200 Increase columns (17040701/29275878/28990944, Cytiva, Marlborough, MA). Finally, proteins were frozen in high salt buffer (50 mM Tris-HCl, pH = 7.5; 1 M NaCl) and stored at −80 °C.