MOESM4 of Enhanced antibody-mediated neutralization of HIV-1 variants that are resistant to fusion inhibitors

Additional file 3: Fig. S4. Spontaneous gp120 shedding from cell surface. The susceptibility of gp41 mutants to spontaneously shed gp120 was determined by flow cytometry and ELISA as described previously [79]. Briefly, culture medium of transiently transfected envelope expressing cells was exchanged for fresh medium containing Brefeldin A (BioLegend) and 0.2 % Sodium azide. Cells were then incubated for 15 h at 37˚C, 5 % CO2. (a) Level of envelope expression before and after incubation was compared by staining with 2G12. (b) Amount of gp120 released during incubation was determined by gp120 capture ELISA. As a positive control, cells expressing WT envelope was incubated with 20 µg/ml sCD4, which trigger gp120 shedding. Cells expressing SIV Env (SIV) and no Env (No Env) were used as negative control. The results are shown as the means ± standard errors of four replicas