Figure 3: Nef does not affect ASK1-mediated Vif inactivation.

(a) Nef inhibits the autophosphorylation of ASK1. HEK293 cells were cotransfected with plasmids encoding XP-ASK1 (100 ng) and Nef-FLAG (100 or 200 ng). Cell lysates were subjected to western blot analysis using the indicated antibodies. (b) Nef does not alter the effect of ASK1 on Vif-mediated A3G degradation. HEK293 cells were cotransfected with plasmids encoding XP-ASK1 (500 ng), HA-A3G (10 ng), Vif (50 or 100 ng) and Nef-FLAG (1 μg). Protein expression was detected by western blot. The numerical values below the blot indicate the amounts of HA-A3G determined with densitometry. (c) HEK293 cells were transfected with the indicated ASK1 mutants (ΔN, constitutively active; K709M, kinase-dead). Cell lysates were subjected to western blotting against the indicated antibodies. (d) HEK293 cells were cotransfected with plasmids encoding HA-A3G (10 ng), Vif (50 or 100 ng) and with wild-type ASK1 or one of its kinase mutants (500 ng). Protein expression was detected by western blot. The numerical values below the blot indicate the amounts of HA-A3G as determined by densitometry. Full images for all western blots analysis are shown in Supplementary Fig. 5.