Figure 6.

 Proteins encoded by partially penetrant RB-1 alleles are defective for E2F binding. (A,C) SAOS2 cells transiently transfected with plasmids encoding the indicated HA-tagged pRB proteins were lysed and immunoprecipitated with an anti-HA antibody. The immunoprecipitates were immunoblotted with an anti-HA antibody (A) or were assayed for coimmunoprecipitated E2F by gel-shift analysis of deoxycholate released proteins by use of a ³²P-radiolabeled E2F DNA-binding site (C). (B) E2F DNA-binding activity in transiently transfected cells. Nuclear extracts were prepared from SAOS2 cells transiently transfected with plasmids encoding the indicated HA-tagged pRB proteins and analyzed in gel-shift assays using an ³²P-radiolabeled E2F DNA-binding site. (Solid arrows) Complexes containing free E2F; (open arrow and solid circle) pRB/E2F and p107/E2F complexes, respectively. (D) E2F complexes in stably transfected cells. SAOS2 cells were transfected with plasmids encoding the indicated HA-tagged pRB proteins along with a neomycin resistance plasmid. Following 2 weeks of selection in media containing G418, nuclear extracts were prepared and analyzed as in B.