Identification of the components of E2F complexes in quiescent human primary CD34⁺ cells by supershift assays. CD34⁺ cells were purified and whole cell extracts prepared and EMSA was performed (see Materials and Methods). Equal amounts of CD34⁺ cell whole cell lysate (8 μg) were incubated with a ³²P-labelled probe containing an E2F binding site and supershift assays were performed by the addition of 2 μL of the appropriate antibody (as indicated for each lane). These were then run on a 4% nondenaturing polyacrylamide gel. Excess unlabelled mutant E2F probe was added to all lanes to ensure specificity of binding. The lanes shown in each panel are from a single gel and the results are representative of four experiments. (A) The E2F complexes are arrowed as band ‘A’ and ‘B’ (lane 1). No supershifts were detected with anti-E2F–1, anti-E2F–2, anti-E2F–3, or anti-pRb antibodies (lanes 2, 4, 5, and 6). A lower complex (starred) appeared with the addition of the E2F-1 antibody and control peptide (lane 2), but was also present with the E2F-1 antibody and E2F-1 peptide (lane 3) and hence was considered to be nonspecific. (B) Both E2F complexes (lane 7) were supershifted by the addition of anti E2F–4 antibody (C-108) (lane 8). Similarly, a proportion of both complexes are retarded with a second E2F-4 antibody (C-20) in the presence of a nonspecific control peptide (lane 9), whereas the supershift is abolished by the addition of a specific E2F-4 peptide (lane 10). No supershift was detected with the addition of anti-E2F–5 antibody (lane 11). (C) A supershift of complex ‘A’ occurs with the addition of anti-DP–1 antibody with control peptide (lane 12) and is overcome by addition of specific DP-1 peptide (lane 13). (D) No supershift occurred with the addition of anti-p107 antibody (lane 15), as compared with the control lane (lane 14). Both E2F complexes were abolished with anti-p130 antibody (lane 15).