Fig 6.

Effects of HIV-1_AD8 Env FPPR changes on processing, subunit association, and gp120-trimer association. HOS cells transiently expressing the wt HIV-1_AD8 Env and the indicated mutants with C-terminal His₆ tags were harvested 72 h after transfection. The cells were lysed, and fractions of the cell lysates were reserved as Input samples. The remainder of the cell lysates was incubated at room temperature for 1.5 h with Ni-NTA beads. The cell supernatants were incubated at room temperature for 1.5 h with GNL beads. Total cell lysates (Input), Ni-NTA precipitates, and GNL precipitates were western blotted with a goat anti-gp120 antibody. HOS cells transfected with the empty pcDNA3.1 vector serve as a negative control. The GNL precipitates from the cell supernatants reflect the level of gp120 shed from the cell-associated Env and were used to calculate subunit association. The Ni-NTA precipitates from the cell lysates measure the relative levels of gp160 and gp120 precipitated in the presence of NP-40 detergent and were used to calculate gp120-trimer association. The results of a typical experiment are shown.