FIG. 1.

Two distinct populations of human Pol-Prim can be distinguished by monoclonal anti-p180 antibodies in vivo. (A) Asynchronously growing CEM cells were metabolically labeled with [³²P]orthophosphate for 2 h (lanes 1 to 4) or with [³⁵S]methionine-cysteine for 1 h (lanes 5 to 8). Equal amounts of protein were used to immunoprecipitate (IP) the Pol-Prim complexes. Immunoprecipitates were separated by SDS-PAGE (8% polyacrylamide) and visualized by autoradiography. (B) Cell lysates (500 μg) from asynchronously growing CEM and CV-1 cells were used to immunoprecipitate Pol-Prim with monoclonal anti-p180 antibodies SJK132-20 (lanes 1 and 5) and HP180-12 (lanes 2 and 6) or normal mouse IgG (mIgG; lanes 3 and 4). Immunoprecipitates were separated by SDS-PAGE (10% polyacrylamide) and analyzed by Western blotting with anti-Pol α monoclonal antibody HP180-7 (1:5). (C) Pol-Prim was sequential immunoprecipitated from CV-1 total-cell lysate (500 μg) four times on SJK132-20–Sepharose beads (lanes 1 to 4) and subsequently two times on HP180-12–Sepharose beads (lanes 5 and 6). Immunoprecipitates were separated by SDS-PAGE (10% polyacrylamide), and the p180 and p70 subunits were detected with monoclonal anti-Pol α antibody HP180-7 (1:5) and anti-p70 antiserum (1:5,000). One hundred micrograms of lysate from CV-1 cells was used as a positive control for the expression of the p180 and p70 subunits of Pol-Prim (lane 7). Pol* indicates a 165-kDa degradation product of the 180-kDa subunit of Pol-Prim.