Figure 5.

 pRB family members differ in flat cell induction. (A) Production of pRB family members in SAOS2 cells. SAOS2 cells transiently transfected with plasmids encoding the indicated HA-tagged wild-type or mutant pRB family members were lysed and immunoprecipitated with an anti-HA antibody. The immunoprecipitates were resolved by electrophoresis in a 7.5% SDS–polyacrylamide gel and immunoblotted with an anti-HA antibody. (B) pRB family members induce a cell cycle block in SAOS2 cells. SAOS2 cells were transiently transfected with plasmids encoding the indicated wild-type or mutant pRB family members along with a plasmid encoding the cell surface marker CD19. Seventy-two hours later the DNA content of CD19-positive cells was determined by FACS. The y-axis indicates the absolute increase in the percentage of cells in G₁ relative to cells transfected with the backbone expression plasmid. Data shown is from a single experiment and is representative of data obtained in three independent experiments. (C) Flat cell induction. SAOS2 cells were transfected with plasmids encoding the indicated wild-type or mutant pRB family members, along with a neomycin resistance plasmid. The number of flat cells per ten 100× fields following 2 weeks of G418 selection was determined by manual counting. Data shown are from a single experiment and is representative of data obtained in three independent experiments.