FIG. 8.

MCM2 interacts exclusively with hypophosphorylated Pol-Prim in G₁/S. Fractions 4 (G₁/S) and 6 (S/G₂) from elutriated CEM cells (Fig. 3A) were immunoprecipitated (IP) with anti-p180 antibodies as indicated. Five hundred micrograms of protein extract was used to precipitate phosphorylated Pol-Prim with SJK132-20. One thousand micrograms of protein extract from G₁/S and 2,000 μg from S/G₂ cells were used to immunoprecipitate hypophosphorylated Pol-Prim with HP180-12. Cell lysates (50 μg) from each fraction were used as positive controls for protein expression (lysate). The precipitates were subjected to SDS-PAGE (8% polyacrylamide) and analyzed by Western blotting. The presence of p180 (Pol) and coimmunoprecipitating MCM2 was detected with monoclonal antibody anti-p180 HP180-7 (1:5) and polyclonal antibody anti-MCM2 (1:200).