Figure 3
BIV Vif interacts with btCul2 and MVV Vif with oaCul5, while CBF-β are not involved. (A-C) 293 T cells (5 × 106) were co-transfected with 5 μg cmyc-tagged BIV Vif (MVV Vif or HIV Vif) and 5 μg VR1012, 5 μg Flag-tagged btCBF-β (oaCBF-β or hCBF-β) and 5 μg VR1012, 5 μg cmyc-tagged BIV Vif (MVV Vif or HIV Vif) and 5 μg Flag-tagged btCBF-β (oaCBF-β or hCBF-β). At 48 h after transfection, cell lysates were co-immunoprecipitated with an anti-cmyc antibody, followed by SDS-PAGE and immunoblot analysis using anti-cmyc and anti-Flag antibodies. (D-F) 293 T cells (5 × 106) were co-transfected with 6 μg HA-tagged BIV Vif (MVV Vif, or pVHL) and 10 μg VR1012, 10 μg cmyc-tagged btCul2 (oaCul2 or hCul2) and 6 μg VR1012, 6 μg HA-tagged BIV Vif (MVV Vif or pVHL) and 10 μg cmyc-tagged btCul2 (oaCul2 or hCul2). Cells were co-immunoprecipitated with HA beads followed by SDS-PAGE and immunoblot analysis using anti-HA and anti-cmyc antibodies. (G-I) 293 T cells (5 × 106) were co-transfected with 4 μg HA-tagged BIV Vif (MVV Vif or HIV Vif) and 8 μg VR1012, 8 μg cmyc-tagged btCul5 (oaCul5 or hCul5) and 4 μg VR1012, 4 μg HA-tagged BIV Vif (MVV Vif or HIV Vif) and 8 μg cmyc-tagged btCul5 (oaCul5 or hCul5). Cells were co-immunoprecipitated with HA beads followed by SDS-PAGE and immunoblot analysis using an anti-HA and anti-cmyc antibodies. All co-immunoprecipitation experiments were repeated three times.