Figure 6.

Sumoylation affects ATF7 activity. (A) ChIP analysis of genomic E-selectin promoter: HeLa-SUMO cells were transfected with px-ATF7WT(lanes 1,3) or px- ATF7K118R (lanes 2,4). Two days later, cells were subjected to ChIP assay with antibodies against ATF7 (lanes 1 and 2) or Flag (lanes 3 and 4). Endogenous E-selectin promoter DNA, coprecipitated with the indicated antibodies, was detected by PCR. (B) Recombinant pSG5-based vectors (0.5 µg) directing the expression of the various chimeras were transfected into HeLa-SUMO cells, and cell lysates were analyzed by EMSA: 20 µg of extracts were preincubated with either 50 ng of non-specific (mt) or specific (wt) competitor before addition of E-selectin CRE probe. Where indicated, reactions were further incubated (30 min, 0°C) with the anti-ATF7 antibody before loading on the gel. The black arrowhead points to the specific complex and the white one to the supershifted one. (C) Recombinant pX-based vectors (0.5 µg) directing the expression of the various chimeras were transfected into HUVEC-C-cells. Two days later, RNA was isolated and E-selectin mRNA expression was analyzed by semi-quantitative RT–PCR method using β-actin as internal control. Ethidium bromide-stained gel is shown in the figure.