Western Blot Analysis of Viral Proteins

All proteins and pseudovirions were run on 4–20% denaturing polyacrylamide gels (Bio-Rad, Hercules, CA) and transferred to nitrocellulose membrane using Trans-blot Turbo (Bio-Rad, Hercules, CA). Membranes were rinsed with PBST, blocked with 5% skim milk in PBST for 1 h followed by probing of GP and p24 with monoclonal antibody (mAb) H3C8 at a dilution of 1:1000, [60 (link)] and mouse mAB B1217M at a dilution of 1:2000 (Genetex, Irvine, CA), respectively. The mAb H3C8 was humanized by cloning its variable heavy and light chain fragments in human IgG expression vectors obtained from Dr. Michel Nussenzwieg (The Rockefeller University). Membranes were washed three times with PBST, incubated with horseradish peroxidase-conjugated anti-human IgG (Invitrogen, ThermoFisher, Waltham, MA) and anti-mouse IgG (ThermoFisher, Waltham, MA) for 1 h at room temperature and developed with SuperSignal West Pico PLUS chemiluminescent substrate (ThermoFisher Scientific, Waltham, MA).