Cullin5 is required for Vif-induced G₂ arrest and its effect is dose dependent. (A) Western blot of HeLa cell lysates at day 3 after transfection with APO3G-HA + Vif vector (lane 1), control vector (lane 2), Vif mutant SLQ (lane 3), or Vif mutant C114S/C113S (lane 4). Expression of Vif and APO3G was measured in cell lysates run on parallel gels; blots were probed with anti–HA antibody (for APO3G expression, upper bands) or with anti–Vif antibody (lower bands). Vif expression was similar among cells transfected with Vif or Vif mutants. Vertical lines have been inserted to indicate a repositioned gel lane. (B) Vif-induced G₂ arrest is dependent on an interaction between Cul5 and Vif. Vertical bars indicate the G₂/G₁ ratio obtained from cell-cycle profiles of HeLa cells transfected with control vector (pcDNA), Vif alone (Vif), or one of the Vif mutants (Vif SLQ or C114S/C113S) at 2-3 days after transfection. The SLQ and C114S/C133S mutants fail to interact with the Cul5-ElongB/C E3 ligase. The mean ± SE of 3 experiments is shown. (C-F) All experiments show results obtained from 3-day cultures of HeLa cells cotransfected with GFP and either VR1012 (control vector) or one of the Cul5 mutants Cul5ΔNedd8 (dominant-negative mutant) or Cul5ΔN1 (negative-control mutant). (C) Proportion of cells in G₂ compared with G₁ after cotransfection with Vif and VR1012, Cul5ΔNedd8, or Cul5ΔN1. The average ± standard error of 3 experiments is shown for each condition. (D) Cell-cycle profiles of Control (column 1) and Cul5 mutant–transfected HeLa cells (columns 2 and 3), marked by the presence (top row) versus the absence (bottom row) of GFP. Vertical axes denote cell number. Horizontal axes denote total DNA content by PI staining. Peaks in each plot denote cells at the G₁ or G₂ phase of the cell cycle as labeled. Each plot shows one representative experiment of 3 conducted for each condition. (E) Dose dependence of the proportion of cells in G₂ compared with G₁ after transfection with decreasing ratios of Vif + control vector or one of the Cul5 mutants. For each condition, 3 ratios were tested: 2:1, 1:1, and 1:2 (horizontal axes). Black bars, GFP⁺ cells; white bars, GPF⁻ cells. Cell-cycle profiles were analyzed by PI staining at 3 days after transfection. The proportion of cells in G₂ compared with G₁ was obtained by analysis of peaks of cell-cycle profiles such as those shown in (B). (F) Western blot of cell lysates 3 days after transfection with control vector (lane 1), the Cullin5 dominant-negative mutant (lane 2), or the Cullin5 control mutant (lane 3). Blot was probed with anti–Cul5 antibody or anti–Vif. Anti–actin antibody confirms that equal amounts of cell extract were run in each lane.