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Figure 5 | Biology Direct

Figure 5

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

Figure 5

Cell cycle regulatory proteins reside in the centrosome during G2,M arrest induced by HIV-1 infection and adriamycin. (A) Western blot analysis of centrosomes isolated from Jurkat cells infected with NL4-3e-n-GFP (HIV; MOI of 2) for two days. Centrosomes were isolated by discontinuous sucrose gradient and fractions were collected and separated by SDS-PAGE and western blotted for Plk1, CyclinB1, γ-tubulin, Cdk1, 14-3-3 θ, centrin, Vif, and Vpr. Lanes 1–6 represent fractions from the bottom of the gradient upward, with centrosomes most abundant in lane 3. Whole cell lysates were run in lane 8 and volume from the top of the gradient equivalent to that used for each fraction was run in lane 7 to demonstrate sedimentation of centrosomal proteins through the gradient. "L" indicates a light exposure and "D" indicates a darker exposure of the chemilumigraph of the middle part of the gel. (B) Viability (top) and DNA content analysis (bottom) by flow cytometry for the culture in (A; HIV (+)) and untreated Jurkats without (-) HIV infection. The percentage of viable cells was determined by propidium iodide (PI) exclusion and high forward scatter and is indicated in the lower right corner. Infection efficiency was measured by GFP expression (inset; gated population) and the percentage is indicated. DNA content was measured by flow cytometric detection of DNA stained with propidium iodide. The GFP-positive population was analyzed for the HIV-infected culture. (C) Jurkat T cells were treated with adriamycin (adr; 0.2 μg/ml; right panel) for two days or grown asynchronously (left panel) and centrosomes were isolated by discontinuous sucrose gradients as in (A). Centrosomes were most abundant in lanes 2–3 (untreated cells) or lanes 3–4 (adriamycin treated cells). Lanes were as described in panel A. (D) Cell cycle flow cytometric analysis of Jurkat cells in (C).

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