Figure 5.

GST–P2P-(753–909) specifically binds Rb1 through the “pocket” domain. (A) Aliquots of K562 total cell lysate (1 × 10⁷ cells per sample) were incubated with the GST leader sequence, or with GST–P2P-(1–332), GST–P2P-(494–688), GST–P2P-(918–1095), or GST–P2P-(753–909). As precipitation controls, aliquots of the K562 lysate were immunoprecipitated with anti-Rb1 antibody IF8 or normal rabbit serum. Bound proteins were separated by electrophoresis in an SDS/7% polyacrylamide gel and transferred to nitrocellulose. An aliquot of the K-562 crude lysate was included as a positive control for Western blot analysis. Proteins were visualized by probing with anti-Rb1 antibodies IF-8 or C15. (B) Binding of GST–P2P-(753–909) to Rb1 is blocked by competition with E1a protein. Aliquots of a K562 lysate were incubated as above with GST or GST–P2P-(752–909) in the presence (+E1a) or absence (−E1a) of purified adenovirus E1a protein. Bound proteins were separated by electrophoresis in an SDS/7% polyacrylamide gel and transferred to nitrocellulose. Proteins were visualized by probing with anti-Rb1 antibodies IF8 or C15.