Figure 6.

DP-4 regulates chromatin-bound E2F-1. (a, i) U2OS cells were transfected with expression vectors (1 μg) encoding HA-DP-1, HA-DP-4 or empty vector (E) as indicated. After 48 h, chromatin was prepared and ChIP analysis performed using anti-HA, E2F-1 or a nonspecific (NS) antibody control, and binding to the E2F-1, Cdc2 and albumin promoters was assessed as described. (a, ii) Quantification of the ChIP analysis in (i) by real-time PCR. (a, iii) Levels of the ectopic proteins, immunoblotted with anti-HA antibody. (b, i) U2OS cells were treated with DP-4 or non-targeting (NT) siRNA (25 nM). After 48 h, chromatin was prepared and ChIP analysis was performed using anti-E2F-1 or NS antibody control as described above. Binding to the E2F-1 (i), Cdc2 (ii) and albumin control (iii) promoter was assessed as described. (iv) Protein levels in cells treated as described in (i) was assessed by immunoblotting with anti-DP-4 and anti-PCNA. (v, vi) Quantification of E2F-1 binding on the E2F-1 and Cdc2 promoters, respectively. (c) U2OS cells were treated with DP-4 or NT siRNA (25 nM). After 48 h, cells were treated with ultraviolet (UV) light (50 J/m²). After 8 h, chromatin was prepared and ChIP analysis performed using anti-E2F-1 or NS antibody control. Binding to the p73, Apaf1 and albumin promoters was assessed