Mass spectrometry and a proteomic screen of Vif-Flag–transfected HeLa cells yield known and novel cellular proteins and RNP partners of Vif. (A) Cell-cycle profiles of HeLa cells transfected with control vector or with Vif-Flag at 3 days after transfection. Note that Vif-Flag, like untagged Vif, causes transfected cells to arrest in G2. Vertical axes denote cell number. Horizontal axes denote total DNA content by PI staining. Peaks in each plot denote different phases of the cell cycle as labeled. One representative experiment is shown. (B) Silver-stained Western blot of immunoprecipitates (with anti–Flag antibody) of cell lysates obtained from cells transfected with Vif-Flag vector (lane 1) or control vector (lane 2). One representative of 3 experiments is shown. The gel shown was silver stained. (C) Western blot of Vif-Flag–transfected HeLa cell lysates immunoprecipitated with anti–Flag antibody. Left lane, control; right lane, Vif-Flag–transfected cells. Equal amounts of cell extract were analyzed using antibodies against Cdk9 and Cul5 and Vif. Cdk9, Cyclin-dependent kinase; Cul5, Cullin5; Vif, viral infectivity factor; WB, Western blot; α, anti-. (D) Relative proportion of cells in S compared with G1 after transfection with Vif and siRNA against its potential binding partners Brd4 and Cdk9. The various vectors used to transfect synchronized HeLa cells are shown across the x-axis. **P < .01 for bar 2 vs bar 1 and for bar 4 vs bar 2; *P < .05 for bar 6 vs bar 2. The mean ± standard error of 3 experiments is shown. Inhibition of Brd4 or Cdk9 with siRNA reduces the Vif-mediated G1-to-S cell-cycle transition in transfected cells. Fold increases were calculated relative to control siRNA and pcDNA cotransfected cells after normalization of the corresponding S/G1 ratio to 1. (E) Western blot of lysates from HeLa cells transfected with control siRNA or siRNA against Brd4 or Cdk9. Lysates were probed with anti–Cdk9 or anti–Brd4 antibody and analyzed at 2 days after transfection. Note how transfection with Cdk9 siRNA or Brd4 siRNA reduced the expression of Cdk9 and Brd4 proteins, respectively.
