Ni nta agarose columns

Recombinant sgp130 was generated using standard molecular cloning techniques. Briefly, the cDNA encoding sgp130 was amplified by RT-PCR from the total RNA prepared with HepG2 cells (forward and reverse primer sequences: 5′-CCCAAGCTTGCCACCATGGGGAAATATCCGCGCAAG-3′, 5′-CCCAAGCTTGCCACCATGGGGAAATATCCGCGCAAG-3′). Following digestion with restriction enzymes BamHI and HindIII, the PCR fragment was cloned into pcDNA3.1 (Invitrogen, CA, USA) for eukaryotic cell expression. Recombinant sgp130 protein was generated by transfecting pcDNA3.1-sgp130 plasmid into CHO cells and purified from the collected cell medium using Ni-NTA-agarose columns (Sango Biotech) following the manufacturer’s instructions. The purified protein was further dialyzed and stored in 10% glycerol at −80 °C.

BL21(DE3) PTD-ompK was grown at 37°C until an OD_600 nm of 0.5 was reached. The recombinant protein expression was induced by 0.5 mmol L⁻¹ isopropylthiogalactoside treatment for 6 h at 25°C. Bacteria were then harvested and lysed by sonication in 50 ml of denaturing lysis buffer. The His-tagged fusion proteins were purified using Ni-NTA-agarose columns (Sango Biotech) that were pre-equilibrated with denaturing lysis buffer containing 20 mmol L⁻¹ of imidazole. Clarified lysates were applied to the columns, and after extensive washing with lysis buffer plus 20 mmol L⁻¹ of imidazole, recombinant proteins were eluted with 250 mmol L⁻¹ of imidazole according to the manufacturer's instructions. Eluted protein solutions were then dialysed and frozen in 10% glycerol at −80°C.

GlPRMT5 and GlPP2C1 sequences from G. lucidum were deposited in GenBank (OP360010 and OP251201, respectively). The expression vectors pColdI-PRMT5, pColdI-PP2C1, pColdI-PP2C1 R99K, pColdI-PP2C1 R493K and pColdI-PP2C1 R99/493K were transformed into E. coli strain BL21 (DE3), protein expression was induced with 500 µM isopropyl-β-D-thiogalactopyranoside (IPTG), and the strains were grown for 16 h at 16 °C. Purification of recombinant proteins was performed using nickel-nitrilotriacetate (Ni-NTA) agarose columns (Sangon, C600033). In addition, the purified His-PP2C1 recombinant protein (Supplementary Fig. 3) was sent to a professionally qualified antibody preparation company for immunization of rabbits (Chemgen Biotech)^{63 (link)}.