Interaction of the Retinoblastoma Protein with Orc1 and Its Recruitment to Human Origins of DNA Replication
Figure 4
Orc1 forms a stable complex with hypo-phosphorylated Rb inside the cells.
(A) Co-immunoprecipitation experiments performed with lysates from asynchronous HeLa cells using the indicated antibodies for immunoprecipitation and western blottings. (B) Immunodetection of endogenous Rb after co-immunoprecitation with exogenous HA-tagged Orc1 in transiently transfected asynchronous HeLa cells. Additional co-immunoprecipitations with Rb and E2F1 proteins in non-transfected and HA-Orc1-transfected HeLa cells were performed as controls. The band marked by an asterisk (*) represents an unspecific band detected with the mouse anti-Rb antibody IF8. (C) Endogenous Rb detected by western blotting after immunoprecipitation with anti-HA peptide antibody in non-transfected, wt HA-Orc1-, and mutant HA-Orc1 LPGRK-transfected U2OS cells. Additional immunoprecipitations for Rb were performed as controls on the same lysates. The band marked by an asterisk (*) represents an unspecific band detected with the mouse anti-Rb antibody IF8. (D) Immunoblotting to visualize the differently phosphorylated forms of endogenous Rb, after immunoprecipitation with anti-Rb and anti-HA peptide antibodies in U2OS cells not transfected or transfected with wt HA-Orc1, as indicated. Orc1 immunoprecipitated hypo-phoshorylated Rb, showing the same apparent mass as that obtained after treatment of total Rb immunoprecipitates with PP2A phosphatase.
