Fig. 1. Human Dnmt1 associates with Rb in vivo. (A) Diagram representing full-length hDnmt1 and Rb pockets. Amino acid numbers for the Rb A, B and C pockets are indicated. Regions associated with specific function are indicated as: PCNA, proliferating cell nuclear antigen binding; RF, replication foci directing, and Zn, zinc binding. N- and C-termini are from amino acid residues 1–1108 and 1122–1616, respectively, for hDnmt1. Conserved motifs are in roman numerals. (B) Methyltransferase activity co-immunoprecipitates with Rb in a recombinant insect cell expression system using poly(dI–dC)·poly(dI–dC) as substrate DNA. Antibodies used for immunoprecipitation are indicated at the bottom. Pre-immune serum is indicated as PI. (C) hDnmt1 co-immunoprecipitates with Rb in recombinant Sf9 cell extracts. Expression of protein is indicated by a ‘+’. Immunoprecipitation with rabbit IgG is abbreviated as IgG. Positive control retinoblastoma (C Rb) and human maintenance methyltransferase (C hDnmt1) are indicated. (D) Left panel: co-immunoprecipitation of hDnmt1 in HEK 293 cell nuclei extracts using antibody against either the N- or C-terminus of Rb. Control rabbit IgG does not bind to hDnmt1. Right panel: association of Rb and Dnmt1 in the presence (+) or absence (–) of serum using C-terminus antibody for Rb. For both panels, HEK 293 cell nuclear extract was used as a control to show expression of Rb as well as hDnmt1 in cells. Cells used for the study (NIH 3T3 and HEK 293) are indicated at the top.