Protein a sepharose column

Mouse forebrain tissues were lysed in a buffer containing 50 mM tris-HCl (pH 7.5), 0.15 M NaCl, 0.1% Triton X-100, 4 mM EDTA, 4 mM EGTA, 1 mM Na₃VO₄, 50 mM NaF, 1 mM dithiothreitol, and protease inhibitors (trypsin inhibitor, pepstatin A, and leupeptin) and centrifuged at 15,000g for 10 min. Supernatants were collected and incubated at 4°C for 4 hours with constant rotation on a Protein A Sepharose Column (GE Healthcare) that had been prebound with His-tagged BG4 (or normal rabbit IgG for control) and anti-6×His. The bound proteins were then washed with tris-buffered saline and eluted with 2.5% acetic acid. The solution was exchanged and concentrated with 50 mM triethylammonium bicarbonate using Amicon Ultra-0.5 and then electrophoresed. The protein samples obtained from the gels were reduced and alkylated and then digested with trypsin. LC-MS/MS analysis of all samples was outsourced to Oncomics Co. Ltd. Proteins identified in the control samples that were pulled down with 10 μg of mouse IgG were subtracted from the identified proteins.

The extracellular domains of CD112R and other PVR-like molecules were cloned and fused into a pMIgV expression vector containing the constant region of mouse IgG2a. Fusion proteins were expressed by transiently transfecting the freestyle HEK293F cells using the polyethylenimine transfection method, and fusion proteins were purified for supernatant using a protein A–Sepharose column according to the manufacturer’s instructions (GE Healthcare).
Mouse anti–human CD112R (clone 2H6; IgG1) was generated from a hybridoma derived from the fusion of SP2 myeloma with B cells from a mouse immunized with human CD112R-Fc. Hybridoma was adapted and cultured in Hybridoma–serum-free media (Life Technologies). Antibodies in supernatant were purified by HiTrap protein G affinity column (GE Healthcare). LEAF purified mouse IgG1 (clone MG1-45) and functional grade human CD112 mAb clone TX31 were purchased from BioLegend. Functional grade human TIGIT mAb (clone MBSA43) was purchased from eBioscience. Human CD226 mAb (clone DX11) was purchased from Abcam. All other antibodies used in flow cytometry were purchased from BD, eBioscience, R&D Systems, or BioLegend.

The selected human PCSK9 scFvs in the phages were amplified by PCR and inserted into a modified mammalian expression vector (pFUSE2-CLIg-hk and pFUSE-CHIg-hG1; InvivoGen). The plasmids were used to transfect 293 cells using the ExpiFectamine™ 293 Transfection Kit (Gibco; Thermo Fisher Scientific, Inc.), and the culture supernatant was collected after 8 days. The antibodies were separated and purified from the supernatant using a Protein A Sepharose column (GE Healthcare Bio-Sciences). The integrity of the antibodies was determined by ELISA and SDS-PAGE.