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Figure 2 | Biology Direct

Figure 2

From: 14-3-3 theta binding to cell cycle regulatory factors is enhanced by HIV-1 Vpr

Figure 2

Increased Cdk1, Cdc25C and CyclinB1 association with 14-3-3 θ but stable nucleocytoplasmic distribution during HIV- and Vpr-induced G2,M arrest. Jurkat cells were infected as in Fig. 1 with RT- NL4-3e-n-GFP virions either with (Vprv) or without (Δ) hVpr supplied in trans or with RT+ NL4-3e-n-GFP (HIV; MOI 2). (A) Cell lysates were harvested two days post-infection for immunoprecipitation with 14-3-3 θ and immunoblotting for CyclinB1, Cdc25C-P.S216, Cdc25C, Cdk1-P.Y15, Cdk1, 14-3-3 θ, and Vpr as indicated. The 14-3-3 θ signal in the IP does not reflect poor immunoprecipitation of 14-3-3 θ but rather the result of membrane stripping prior to 14-3-3 θ blotting. (B) DNA content analysis (y-axis) is shown in flow cytometric dot plots against GFP (x-axis) on the right and as a histogram in the inset for the samples in (A). Note that the y-axis of the parent graph becomes the x-axis of the inset graph. The quadrant gate demarcates approximate G1 (lower) and S/G2,M (upper) populations and the percentage of cells in each relevant quadrant is indicated. The DNA histogram profile analysis was separated into GFP-positive (+) and negative (-) populations by the x-axis gate for the HIV-infected culture; as expected the infected cells (+) show G2 arrest, but the uninfected cells (-) are mostly G1. (C) G2,M cell cycle arrest caused by Vprv and HIV infection does not alter the cytoplasmic and nuclear distribution of 14-3-3 θ, Cdc25C, Cdk1, and CyclinB1. Jurkat T cells shown in (A-B) that were infected with NL4-3e-n-GFP RT- Δ Vpr (Δ), RT- wt Vpr (Vprv), or NL4-3e-n-GFP RT+ (HIV) for two days were lysed and biochemically separated into cytoplasmic and nuclear fractions. Lysate fractions were blotted as in (A) (the lower panel of Vpr blot represents a longer exposure in which Vprv is more apparent), with the addition of probes for HIV-1 Vif, Poly(ADP-ribose) polymerase (PARP) as a nuclear marker, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a cytoplasmic loading control. Cell cycle profiles and GFP expression are shown in (B). (D) Viral lysates (20 μg) of RT- NL4-3e-n-GFP virions with (+) or without (-) Vpr were western blotted for CyclinB1, Cdk1, p24, and Vpr as indicated.

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