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

Additional file 1:

Biochemical analysis of cell cycle protein subcellular distribution. The data represent the nucleocytoplasmic distribution of Cdk1, Cdc25C and CyclinB1 during HIV- and Vpr-induced G₂,M arrest. (A) Jurkat cells shown in Fig. 2A–B that were infected with NL4-3_e-n-GFP RT- Δ Vpr (Δ), RT- wt Vpr (Vpr_v), or NL4-3_e-n-GFP RT+ (HIV) for two days were lysed and biochemically separated into cytoplasmic and nuclear fractions. Lysate fractions were blotted as in Fig. 2A (the lower panel of Vpr blot represents a longer exposure in which Vpr_v 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 Fig. 2B. (B) Viral lysates (20 μg) of RT- NL4-3_e-n-GFP virions with (+) or without (-) Vpr were western blotted for CyclinB1, Cdk1, p24, and Vpr as indicated.

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Additional file 2:

Biochemical analysis of cell cycle proteins in chemically cross-linked cells. The data depict CyclinB1 accumulation in high molecular weight complexes following Vpr_v G₂,M arrest. (A-C) Jurkat cells treated with RT- NL4-3_e-n-GFP to deliver wild-type (lane 2, "w"), R80A (lane 3, "A") or no (lane 1, Δ) Vpr for two days were cross-linked with disuccinimidyl suberate (DSS, 1 mM). Whole cell lysates were separated by 3–8% Tris-Acetate SDS-PAGE and transferred to membranes that were blotted for CyclinB1 (A; 55 kD) and Cdk1 (B; 34 kD). High molecular weight bands of interest are indicated with, "a," "b," "c," and "d." (C) Flow cytometric histograms of the DNA content analysis for samples in (A-B). (D-F) Jurkat cells mock-infected (lane 1, "m") or infected with RT- (Vpr_v; lane 2, "V") or RT+ (HIV; lane 3, "H") NL4-3_e-n-GFP for two days were analyzed as in (A-C) for DNA content (D) and higher-order CyclinB1 complex formation by DSS cross-linking (E). Propidium iodide DNA staining is plotted against GFP, and the inset histograms depict the DNA content for the entire culture or the GFP positive (+) and GFP negative (-) subsets for the HIV-infected culture. Note that the y-axis of the parent graph is represented by the x-axis in the inset graph. The percentage of cells in the GFP+ gate is indicated. (E) Western blot analysis of untreated control (-) and DSS cross-linked (+) cells for CyclinB1 (top) and Cdk1 (bottom). No higher molecular weight bands were seen in the Cdk1 blot due to membrane stripping following the CyclinB1 blot. The same membrane was stripped and reprobed for the α-catalytic subunit of PKA (F; 40 kD) as a positive crosslinking control.

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