Figure 1: ASK1 binds and counteracts Vif.

(a,b) ASK1 inhibits Vif-mediated A3G degradation. HEK293 cells were cotransfected with plasmids encoding Vif (250 ng), GFP-A3G (50 ng) and the indicated HA-tagged MAP3Ks or Xpress (XP)-tagged ASK1 (500 ng). After 48 h, GFP intensities (n=3, mean±s.d.), cell images and protein expression levels were analysed by flow cytometry (a), fluorescence microscopy and western blotting (b), respectively. Scale bar, 100 μm. (c) ASK1 interacts with Vif in cells. Cells were cotransfected with plasmids encoding ASK1-HA and Vif. Cell lysates were immunoprecipitated with an anti-HA antibody and the bound proteins were then analysed by western blotting. (d) In vitro interaction of ASK1 with Vif. Schematic representation of the amplified luminescent proximity homogeneous (AlphaScreen) assay used to detect the direct protein–protein interaction (left). In this study, FLAG-tagged Vif proteins were co-expressed with CBFβ to stabilize the conformation of the Vif protein^28,63. AlphaScreen analysis (n=3, mean±s.d.) using recombinant ASK1, A3G and Vif proteins is shown (right). GST and DHFR (dihydrofolate reductase) were used as negative controls. ***P<0.001, two-tailed unpaired t-test. (e) HEK293 cells were cotransfected with plasmids encoding ASK1-HA and Vif or one of its truncated mutants (top). Cell lysates were immunoprecipitated with anti-HA antibody and bound Vif proteins were detected with western blot analysis (bottom). (f) HEK293 cells were cotransfected with plasmids encoding Vif and ASK1-HA or one of its truncated mutants (top). Cell lysates were immunoprecipitated with anti-HA antibody and bound Vif proteins were detected by western blot analysis (bottom). Full images for all western blots analysis are shown in Supplementary Fig. 5.