Western blot analyses of the DNA pull-down proteins from HUVEC and HEK293 cells and supershift of the gel mobility complex. (A) DNA pull-down experiment was performed exactly as described for Figure 5. The eluted and flow-through polypeptides were analyzed on SDS-PAGE gel followed by transfer to a nylon membrane and incubation with anti-histone H1 antibody as described in “Materials and methods.” Purified histone H1 was used as positive control (shown in lane 1). Lanes 2 and 3 represent flow-through proteins that were not bound to HH2 probe. Lanes 4 and 5 represents eluted proteins bound to HH2mut probe. Lanes 6 and 7 represent eluted proteins bound to HH2wt probe. White arrow represents the endothelial-specific protein that is recognized by anti-histone H1 antibody. Position of protein molecular weight markers are shown by black arrows. (B) Nuclear extracts prepared from HUVECs (lanes 3-5) were incubated in the absence (lane 3) and presence of 1 μg anti-histone H1 (lane 4) or IgG (lane 5) antibodies prior to addition of oligonucleotide probe (sequences +155 to +184). The anti-histone H1 antibody used was a mouse monoclonal antibody (clone AE-4), and the IgG used was normal mouse IgG. Nuclear extracts from HEK293 cells (lane 2) were used as control to distinguish the position of endothelial-specific complex from other complexes. Lane 1 represents probe only. The positions of the endothelial specific-complex (ESC), and supershifted complexes are shown by arrows and bracket, respectively.