Figure 6.

EloB promotes CBF-β binding to Vif mainly by its C-terminus. (A) Both Flag-tagged EB and EBΔC34 co-immunoprecipitated with EC-HA. 293 T cells (10⁶) were co-transfected with 1 μg of VR-EC-HA and 600 ng of VR-Flag-EB or 3 μg of VR-Flag-EBΔC34. Cell lysates were immunoprecipitated with anti-HA antibody, followed by SDS–PAGE and analyzed by Western blotting with anti-HA, anti-Flag and anti-tubulin antibodies. (B) Over-expression of Flag-tagged EBΔC34 impaired the interaction between Vif and CBF-β. 293 T cells (10⁶) were transfected with 2 μg of VR-Vif-HA and 400 ng of VR-Flag-EB-HA or 2 μg of VR-Flag-EBΔC34. Cell lysates were immunoprecipitated with an anti-HA antibody, followed by SDS–PAGE and immunoblot analysis. (C) The PPL → AAA mutation of Vif resulted in impaired CBF-β binding to Vif. 293 T cells (10⁶) were transfected with 800 ng of VR-Vif-HA or 3 μg of VR-VifΔPPL-HA or VR1012. Cell lysates were immunoprecipitated with an anti-HA antibody, followed by Western blotting with anti-HA, anti-CBF-β, anti-EloB and anti-EloC antibodies. The result is representative of three independent experiments.