FIG. 8.

Analysis of cell surface d8 Env expression by biotinylation. CEM (12D-7) cells were infected with VSV-G-pseudotyped NL4-3 KFS (env minus), wt NL4-3, or NL4-3/d8. Cells were biotinylated with sulfo-N-hydroxysuccinimide–biotin; cell lysates were immunoprecipitated with AIDS patient sera (to detect total Env expression) or precipitated with neutravidin-agarose beads (to detect cell surface Env) and were subjected to Western blotting with rabbit anti-gp120 polyclonal antibody. To confirm that only cell surface Env was biotinylated, we treated cells infected with VSV-G-pseudotyped NL4-3 with brefeldin A (BfrA) (Materials and Methods).