The antibody recognizes E2F7 isoform 2 (E2F7-A), but not E2F7 isoform 1 (E2F7-B). 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-E2F7-A antibody. Peroxidase-conjugated anti-rabbit antibodies followed by ECL were then used. As shown in panel B, the antibody recognizes E2F7-A, but not E2F7-B.
The antibody immunoprecipitates endogenous E2F7-A. 500 ug of whole cell extracts from K562 cells were immunoprecipitated with 2 ul of the crude rabbit serum or the corresponding pre-immune serum (PI). Immunoprecipitated proteins were fractionated on 8% SDSPAGE and transferred to nitrocellulose membranes. The membranes were then probed with a previously described anti-E2F7 antibody (1). Peroxidase-conjugated anti-rabbit antibodies followed by ECL were then used. The arrow points to immunoprecipitated E2F7-A.
The anti-E2F7-A antibody is able to supershift a DNA-protein complex that contains E2F7-A. Complex formation employing a radiolabeled E2F element from the human DHFR promoter and nuclear extracts from mock transfected Saos-2 cells (-) or nuclear extracts from Saos-2 cells transfected with pcDNA3-HAE2F7- A (+) were analyzed by EMSA. Extracts were preincubated in the absence (None) or in the presence of an antibody to the HA tag or the anti-E2F7-A antibody. The arrow indicates the position of the HA-E2F7-A containing complex, whereas arrowheads indicate the position of supershifted complexes.