SDS-PAGE and western blot analysis

For SDS-PAGE, proteins were prepared in 2×-Laemmli sample buffer (Bio-Rad, Philadelphia, PA) added with 5% β-mercaptoethanol and denatured by heating at 95 °C for 5 min. The denatured protein samples (20 µl) were loaded onto 12% precasted polyacrylamide gels (Bio-Rad). For western blot analysis, SDS-PAGE-run samples were then transferred into the nitrocellulose membrane using Trans-Blot Turbo Transfer Packs (Bio-Rad). Then the membrane was blocked with PBS containing 0.5%Tween-20 (PBS-T) and 3% BSA, and incubated with anti-His tag antibodies (Catalogue # MA1-21315, Invitrogen; diluted 1:5,000 in PBS-T) or murine anti-VirGα immune sera (pooled from mice immunized with VirGα protein; diluted 1:1000 in PBS-T) overnight at 4 °C. The membrane was washed with PBS-T and incubated with Horseradish Peroxidase (HRP)-labeled goat anti-mouse IgG (Catalogue # 5220-0460, KPL SeraCare, Gaithersburg, MD) diluted 1:10,000 in PBS-T for 1 h at RT. The membrane was again washed with PBS-T, revealed with Immobilon Western Chemiluminescent HRP Substrate (Millipore Sigma, Burlington, MA) and analyzed using Gel Doc imaging system (Bio-Rad). Blots and gels derived from the same or side-by-side experiments were processed together. The uncropped and unprocessed images used to generate Fig. 1b–d are shown in Supplementary Fig. 3.