PMC

Colony suppression assay. SaOS2 cells were transfected with the parental vector alone (lane 1) or with the different RB cDNAs as indicated (lanes 2–6), and colonies were scored at 3 weeks in the presence of G418 as described in Materials and Methods.

In vivo phosphorylation of stable RB transfectants. Sequential α-RB immunoprecipitation and immunoblot analysis showing lysates containing the 706F mutant and wild-type RB product (lanes 1 and 2). Protein lysates from stable transfectants of the H2009 cell line expressing the parental vector alone (lane 3) or the different RB cDNAs without ectopic cyclins are indicated (lanes 4–7).

In vitro E2F1 binding. [³⁵S]methionine-labeled in vitro-translated RB cDNAs were subjected to SDS/PAGE and autoradiography before (lanes 1, 4, 7, 10, and 13) or after (lanes 2, 5, 8, 11, and 14) precipitation by GST or GST-E2F1 (lanes 3, 6, 9, 12, and 15).

In vivo phosphorylation after transient cotransfection of RB and cyclins. Sequential α-RB immunoprecipitation and immunoblot analysis of protein lysates from the RB(−) tumor line H2009 after transient transfection with either the parental mammalian expression plasmid (lane 1) or the plasmid containing wild-type (wt) RB (lanes 2–6), Δ480 (lanes 7–11), Δex21 (lanes 12–16), Δex22 (lanes 17–21), or Δex4 RB (lanes 22–26). The H2009 cells were cotransfected with different cyclin constructs as indicated, and protein lysates were harvested at 72 hr for analysis.