Co-immunoprecipitation and Western Blot Assays

The co-immunoprecipitation (Co-IP) and western blot (WB) assays were carried out as described previously (59 (link), 60 (link), 63 (link)–69 (link)). In brief, HEK293T cells were seeded on 10 cm petri-dish (Corning) and incubated to ~80% confluence, then cells were co-transfected with 20 μg of plasmid combinations tagged with Myc, Flag, or HA. At 24 h post-transfection, cells were harvested and lysed on ice with 800–1,000 μl RIPA lysis buffer (Beyotime Biotechnology) for 30 min. After that, cell lysates were divided into two parts, 10% lysates were directly prepared as the lysates sample, and 90% lysates were incubated with the indicated Ab (anti-Flag or anti-HA) or nonspecific control mouse antibody (IgG) at 4°C for 6 to 12 h, then the antibody-containing lysates were incubated with 50 μl slurry of protein A/G PLUS-Agarose (Santa Cruz) at 4°C overnight. The bead complex was washed at least three times with 1 ml of PBS. Finally, cell lysates and bead protein complex were subjected to WB analysis to detect the potential interaction of virus-host proteins. The original WB results were shown in the section of Supplementary Material.