Chim3 affects WT-Vif-Cul5 interaction. (A) HEK-293T cells transduced with equal number (5 copies per cell) of empty-, WT-Vif–, and Chim3-LVs were either transfected with Tat and Rev plasmids or left untransfected. At 48 hours after transfection, cells were analyzed for cell-cycle distribution by determining the intracellular PI content. Results are means ± SEM of n = 4. (B) Aliquots of the cells of panel A were used to determine the expression of Vif in the absence or presence of Tat/Rev proteins as indicated. (C-D) In the left panels, 40 μg of whole-cell extracts (Input; WCE) derived from HEK-293T cells transfected with a fixed amount of Cul5-HA plasmid DNA (670 ng/106 cells) and increasing amount of either WT-Vif-Myc (C) or Chim3 DNA vectors (D; lanes 1 and 7 = 80 ng/106 cells, lanes 2 and 8 = 170 ng/106 cells, lanes 3 and 9 = 330 ng/106 cells, lanes 4 and 10 = 670 ng/106 cells, and lanes 5 and 11 = 1330 ng/106 cells) were analyzed by Western blot to determine the relative amount of Cul5 and Vifs. In the right panels, 500 μg of the same WCEs were coimmunoprecipitated with the anti-HA Abs and then immunoblotted with either the anti-HA or the anti-Vif Abs, as indicated. Lanes 6 and 12, containing the same amount of WT-Vif-Myc and Chim3 as lanes 5 and 11, but no Cul5-HA, correspond to negative controls. (E) Competition experiments were performed using HEK-293T cells transfected with a fixed amount of Cul5-HA (670 ng/106 cells) and WT-Vif-Myc (170 ng/106 cells) and increasing amounts of untagged Chim3-expressing vector (lanes 2 and 9 = 80 ng/106 cells, lanes 3 and 10 = 330 ng/106 cells, lanes 4 and 11 = 670 ng/106 cells, and lanes 5 and 12 = 1330 ng/106 cells). Coimmunoprecipitations were carried out using the anti-HA Abs, and the blots were revealed with the anti-HA– and the anti-Vif–specific Abs. Lanes 7 and 14, containing the same amount of WT-Vif-Myc as lanes 6 and 13, but no Cul5-HA, correspond to negative controls. For the left and right panels, see panels C and D.
