The antibody recognizes E2F7 isoform 1 (E2F7-B), but not E2F7 isoform 2 (E2F7-A). Whole cell extracts (10 ug) from Saos-2 cells transfected with either mock pcDNA3 (None), pcDNA3-HA-E2F7-A (E2F7-A), or pcDNA3-HAE2F7-B (E2F7-B) were fractionated on an 8% SDS-PAGE, transferred to nitrocellulose membranes, and probed with a previously described anti-E2F7 antibody (Control) (1) or the anti-E2F7antibody. Peroxidase-conjugated anti-rabbit antibodies followed by ECL were then used. As shown in panel B, the antibody recognizes E2F7-B, but not E2F7-A.
The anti-E2F7-B antibody is able to supershift a DNA-protein complex that contains E2F7-B. Complex formation employing a radiolabeled E2F element from the human DHFR promoter and nuclear extracts from Saos-2 cells transfected with pcDNA3-HAE2F7- B (+) were analyzed by EMSA. Extracts were preincubated in the absence (None) or in the presence of an antibody to the HA tag, anti-E2F7 antibody, or the corresponding pre-immune serum (PI). The arrow indicates the position of the HA-E2F7-B-containing complex, whereas arrowheads indicate the position of super-shifted complexes.