FIG. 3.

CD4-induced shedding of gp120 from JR-FL cell surface Env spikes. (A) The gp120 present in the cell supernatants (100 μl) derived from the JR-FL Env-expressing cells treated with increasing concentration of sCD4 (1–50 μg/ml) was immunoprecipitated by the anti-V3 monoclonal, 39F. The gp120 glycoproteins were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) and the Western blot was developed by polyclonal rabbit anti-gp120 primary antibody followed by horseradish peroxidase (HRP)-conjugated goat antirabbit IgG secondary antibody. Untreated cells were used to evaluate the spontaneous shedding of gp120 from the cell surface spikes. Purified gp120 was used as a positive control (far right lane). The bands on the film were quantitated using the Quantity one software (Bio-Rad) and are plotted in the right panel. (B) The same set of cell supernatants was analyzed by ELISA to detect the presence of shed gp120 as shown. The gp120 present in the samples was captured on the ELISA plate precoated with lectin. Lectin-captured gp120 was then detected using the 39F monoclonal antibody.