Figure 5
Dominant-negative btCul2 and oaCul5 mutants block the degradation, while TPEN can inhibit btA3Z2-Z3 degradation. 293 T cells (0.5 × 106) were co-transfected with (A) 30 ng of HA-tagged btA3Z2-Z3 and 200 ng cmyc-tagged BIV Vif or VR1012, adjusted to 500 ng with 300 ng cmyc-tagged btCul2ΔNedd8, a control vector cmyc-tagged hCul1K720R or VR1012, (B) 15 ng HA-tagged oaA3Z2-Z3 and 200 ng cmyc-tagged MVV Vif or VR1012, adjusted to 500 ng with 300 ng cmyc-tagged oaCul5ΔNedd8, a control vector cmyc-tagged hCul1K720R or VR1012, (C) 50 ng HA-tagged hA3G and 600 ng cmyc-tagged HIV Vif or VR1012, adjusted to 900 ng with 300 ng cmyc-tagged btCul5ΔNedd8, a control vector cmyc-tagged hCul1K720R or VR1012. At 48 h after transfection, the cells were harvested for Western blotting using anti-HA, anti-cmyc and anti-tubulin antibodies. The percentages of btA3Z2-Z3, oaA3Z2-Z3 or hA3G in the presence of btCul2ΔNedd8, oaCul5ΔNedd8 or hCul5ΔNedd8 relative to that in the absence of BIV/MVV/HIV Vif (set to 100%) were calculated. 293 T cells (0.5 × 106) were co-transfected with (D) 30 ng HA-tagged btA3Z2-Z3 and 200 ng cmyc-tagged BIV Vif or VR1012, (E) 15 ng HA-tagged oaA3Z2-Z3 and 200 ng cmyc-tagged MVV Vif or VR1012. After 36 h of transfection, the cells were treated with TPEN at 3.5 μM or DMSO as a control. At 48 h after transfection, the cells were harvested for Western blotting using anti-HA, anti-cmyc and anti-tubulin antibodies. Percentages of degradation with DMSO or TPEN treatment were calculated.