Fig 5.

wtE2F-1 and E2Ftr expression resulted in reduced binding of DREAM to the Hrk gene and was correlated with increased HRK up-regulation and apoptosis. SK-MEL-2 and A375 cells were infected as indicated. After 24 hrs of infection, nuclear and cytoplasma protein were extracted. (A) Nuclear proteins (4 μg) were subjected to EMSA using biotin-labelled DRE-Hrk. PCNA Western blot analysis was used as loading control of nuclear proteins from each sample (Ctrl: control virus infected cell nuclear protein lysate; arrow: supershifted band; arrowhead: specific DREAM-Hrk binding). (B) 50 μg of cytoplasma proteins were subjected to Western blot analysis using HRK, wtE2F-1 or E2Ftr, and α-actin antibody. (C) Supershift assay using 10 μg of control virus-infected cell nuclear protein lysate is shown. The specific binding band of DREAM-Hrk (arrowhead) was shifted upward by the addition of anti-DREAM antibody (arrow), but not by control IgG.