FIG. 6.

Loss of function of the RB 13S-661 mutant cannot be rescued by fusion with the E2F DNA-binding domain. (A) E2F-RB mutant fusion constructs. An E2F-1 fragment from amino acids (aa) 1 to 368 containing the E2F DNA binding domain was fused in frame to RB amino acids 379 to 928 containing either the 661 mutation (DBD-661) or the 661 mutation plus the 13S C-pocket mutation (DBD–13S-661). The E2F-1 DBD and DBD-A/B have been previously described (21). (B) Fusion of an E2F DNA binding domain does not enhance the formation of flat cells. DBD, RB, DBD-A/B, DBD-661, 661, DBD–13S-661, 13S-661, or pCMVneo was cotransfected with a puromycin resistance plasmid into Saos-2 cells. Cells were selected for 7 days in medium containing puromycin, and flat cells were counted. The number of flat cells formed for each transfectant was expressed as a percentage of the number of flat cells formed by RB, which was considered 100%. Results shown are the means and standard deviations of results from three independent experiments. ø, vector-transfected cells.