Figure 3

Vif-induced abnormalities in CyclinB1 and PLK1. Jurkat cells were synchronized and infected as in Figure 1 with the GFP-expressing viruses. These data are representative of three experiments with infection efficiencies ranging from 85-95% based on GFP expression. (A) CyclinB1 localizes to the nucleus in Vif-expressing cells, but is not degraded normally. Subcellular localization of CyclinB1 and Vif was determined by immunofluorescent confocal microscopy as in Figure 2 panel A using a mouse anti-Vif antibody (ARRRP) [54–56] and a rabbit anti-CyclinB1 antibody (Santa Cruz Biotechnology). (B) At least 350 cells were counted from representative fields, and the percentage of cells showing either a degraded, nuclear, or cytoplasmic phenotype for CyclinB1 were plotted at each time point. (C) CyclinB1 degradation is not observed in infected cells, and PLK1 expression is elevated in infected cells. A duplicate blot from Figure 2 panel C was probed with a mouse anti-CyclinB1 antibody (Cell Signaling Technology). PLK1 expression was examined using a mouse anti-PLK1 antibody (Upstate/Millipore). The expression of β-actin using a mouse-anti β-actin antibody (anti-β-actin, Sigma-Aldrich) is provided as a loading control.