Figure 5
Overexpression of DDB1 induces G 2 cell cycle arrest by Vpr and C81. (A) HeLa, HepG2, and 293 T cells were transfected with pME18Neo encoding Flag-tagged wild-type Vpr or C81, or the control pME18Neo-Flag, together with pSV-β-galactosidase. At 48 h post-transfection, the cells were collected and lysed. Lysates with equal β-galactosidase activity were subjected to Western blotting with either a DDB1 specific antibody or the Flag-specific MAb M2. (B) HeLa, HepG2, and 293 T cells were transfected with pME18Neo encoding Flag-tagged wild-type Vpr or C81, or the control pME18Neo-Flag, together with the pDDB1-IRES-GFP expression plasmid and pSV-β-galactosidase. Lower panel: 48 h post-transfection, cells were fixed and stained with PI. GFP-positive cells were analyzed by flow cytometry using CELL Quest for acquisition and ModFit LT for quantitative analysis of DNA content. Arrows indicate the peaks of cells at the G1 and G2/M phases. The G2/M:G1 ratio is indicated at the upper right in each graph.