Figure 4.

Interaction of the DNA ligase I–PCNA complex with GST-p21 beads. (A) DNA ligase I (180 ng) and PCNA (60 ng) were preincubated before incubation with either GST or GST-p21 beads as described in Materials and Methods. Proteins specifically bound to the beads or remaining in the supernatant were detected by immunoblotting with the polyclonal DNA ligase I antiserum and the PCNA monoclonal antibody after denaturing gel electrophoresis. DNA ligase I and PCNA are indicated on the right. (B) DNA ligase (1 μg) and PCNA (0.5 μg) were preincubated as indicated and then immunoprecipitated with DNA ligase I antiserum as described in Materials and Methods. PCNA was detected in the immunoprecipitates by immunoblotting with the PCNA monoclonal antibody. (C) Equal amounts of the immunoprecipitated DNA ligase I–PCNA complex were incubated with GST (1 μg) or GST-p21 (1 μg). The protein A beads were removed by centrifugation, and the supernatants were incubated with glutathione beads. After separation of proteins bound to beads by SDS/PAGE, PCNA was detected by immunoblotting with the PCNA monoclonal antibody. Protein A beads incubated with: lane 1, GST-p21 and lane 2, GST. Glutathione beads incubated with: lane 3, GST-p21 supernatant and lane 4, GST supernatant. The positions of prestained molecular mass standards (Novex) are indicated on the left.