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. 2010 Jun 18;18(1):122–132. doi: 10.1038/cdd.2010.70

Figure 1.

Figure 1

DP-4 is DNA damage inducible. (a) Endogenous DP-4 in HEK293, HeLa, MCF7 and U2OS cells: cell extract (100 μg) prepared from each cell type was immunoblotted with peptide-purified anti-DP-4 antibody; lamin B served as the loading control. (b) U2OS cells were transfected with expression vector encoding HA-DP-4 (1 μg). Immunostaining was performed with anti-HA11 or peptide-purified anti-DP-4 antibody as described; DAPI was used to visualize nuclei. (c) MCF7 cells were treated with ultraviolet (UV) light (50 J/m2). After 8 h, cells were fractionated into cytoplasm and nuclei, and thereafter immunoblotted with anti-DP-4 or anti-nucleophosmin as indicated, the latter serving as a control for nuclear fraction. (d, i) U2OS cells were treated with doxorubicin (2 μM) for 2, 5, 8 and 24 h and extracts immunoblotted with anti-DP-4 antibody; PCNA served as the loading control. (d, ii) U2OS cells treated with etoposide (10 μM) for 2, 4, 6 or 8 h and extracts immunoblotted with the indicated antibodies; PCNA served as the loading control. (d, iii) U2OS cells were treated with ultraviolet (UV) light (50 J/m2), extracts prepared at 2.5, 5.0 and 7.5 h and immunoblotted with anti-DP-4; PCNA served as the loading control. (e, i) HeLa cells were treated with etoposide (10 μM) for 2, 5, 8 and 24 h and extracts immunoblotted with anti-DP-4 antibody; PCNA served as the loading control. (e, ii) HeLa cells were treated with doxorubicin (2 μM) for 2, 4, 6 or 8 h and extracts immunoblotted with the indicated antibodies; PCNA served as the loading control. (e, iii) HeLa cells were treated with ultraviolet (UV) light (50 J/m2), extracts prepared at 2.5, 5.0 and 7.5 h and immunoblotted with anti-DP-4; PCNA served as the loading control. (f) U2OS cells were transfected with HA-DP-4 (1 μg), and 44 h later cells were treated with etoposide (10 μM) and left for 4 h, harvested, immunoprecipitated with anti-HA11 or a nonspecific (NS) antibody and the immunoprecipitates immunoblotted with anti-E2F-1 and HA11 (DP-4) antibody. DP-4 is indicated by the arrow, together with the NS IgG heavy chain. (g) U2OS cells were treated with etoposide (10 μM), left overnight, harvested and immunoprecipitated with anti-E2F-1 (KH95) or NS antibody. The immunoprecipitates were immunoblotted with anti-E2F1 and anti-DP-4