Characterization of the Interaction of Full-Length HIV-1 Vif Protein with its Key Regulator CBFβ and CRL5 E3 Ubiquitin Ligase Components
Figure 5
Vif-CBFβ-EloB/C is an RNA-independent stable complex in solution.
(A) Vif-CBFβ-EloB/C was not dependent on RNA. The purified complex (2 mg/ml, 100 µl) was incubated with 2 µl RNase Mix (RNase A/T1 Mix, Catalog EN055, Fermentas) at 37°C for 4 h according to the manufacturer's protocol, followed by buffer exchange to remove the EDTA. The complex then was analyzed by His-tag affinity pull-down. (B) The Vif-CBFβ complex is not stable. Purified Vif-CBFβ complexes were concentrated to 4 mg/ml and, after clarification at 13,000 g for 10 min, the supernatants were stored at 4°C (Input). Samples were then removed at different times (0 h, 6 h, 24 h), and after clarification at 13,000 g for 10 min, the supernatants (S) were removed and the pellets (P) resuspended to the original volume and checked by SDS-PAGE. (C) Purified Vif complexes were concentrated to 5 mg/ml (Input) and stored at 4°C overnight (∼16 h). The supernatants (S) were removed after clarification at 13,000 g for 10 min, and the pellets (P) were resuspended to the original volume, then checked by SDS-PAGE.