Figure 2

EloB is critical for the interaction between Vif and CBF-β. (A) EloB did not affect the interaction between Vif and A3G. 293 T cells (10⁶) were transfected with the indicated siRNAs. At 24 h after siRNA transfection, cells were co-transfected with 2.5 μg of VR-Vif and 1 μg of VR-A3G-HA or VR1012. The cells were treated with MG132 (10 μM) for 12 h before harvesting. Cell lysates were co-immunoprecipitated (Co-IP) with anti-HA antibody, followed by SDS–PAGE and immunoblot analysis. (B) EloB silencing impaired both the Vif-CBF-β and the Vif-Cul5 interactions. SiRNA-treated cells (10⁶) were transfected with 2.5 μg of VR-Vif-HA or VR1012. Cell lysates were immunoprecipitated (IP) with anti-HA antibody, followed by SDS–PAGE and immunoblot analysis. (C) Endogenous CBF-β co-immunoprecipitated with Vif–HA but not EloB–HA (EB-HA). 293 T cells (10⁶) were transfected with 2.5 μg of VR-Vif-HA or VR-EloB-HA or VR1012. Cell lysates were immunoprecipitated with an anti-HA antibody, followed by SDS–PAGE and immunoblot analysis. (D) CBF-β, EloB (EB) and EloC (EC) co-immunoprecipitated with Vif–Myc but not Vif SLQ–Myc. 293 T cells (10⁶) were transfected with 2 μg of VR-Vif-Myc, 2.5 μg VR-Vif SLQ-Myc or VR1012. Cell lysates were immunoprecipitated with an anti-Myc antibody, followed by SDS–PAGE and immunoblot analysis. All results are representative of three independent experiments.