FIG. 4.

Agents that alter the myristoyl-CoA pool modulate the Nef-NMT-1 complex. (A) Cos7 cells coexpressing T7.NMT-1 and T7.NMT-2, either alone (lanes 1 and 6), or together with wild-type (wt) (NA7.AH) (lanes 2 to 5 and 7) or myristoylation-defective [NA7_(G2A).H] (lane 8) HIV-1 Nef were cultured in the presence of 50 mM lauric acid (lane 3), 50 mM myristic acid (lane 4), 50 mM palmitic acid (lane 5), or 5 μM Triacsin C (lanes 6 to 8) or left untreated (lane 1). Nef-associated NMT was immunoprecipitated from detergent extracts with α-H MAb (IP:α-H). Immunoprecipitates and aliquots of detergent extracts were resolved by SDS-PAGE, and NMT-1 and NMT-2 were detected by immunoblotting with T7 epitope-specific MAb (WB:α-T), whereas Nef was detected with H epitope-specific MAb (WB:α-H). (B) Nef-associated NMT-1 and NMT-2 were quantitated by densitometry scanning of chemiluminescence images such as those shown in panel A. Mean values and standard deviations from three independent experiments are shown. Data were normalized to the NMT-1 associated with the NA7.AH Nef in untreated cells (lane 2).