FIG. 3.
Baculovirus p33 forms a stable complex with human p53. SF-21 cells were transfected with plasmids expressing the indicated combinations of Flag- and Epi-tagged genes under Phsp70 control (indicated by plus signs). Transfected cells were further mock or AcMNPV infected (indicated by plus signs adjacent to AcMNPV) and heat shocked 6 h following infection. At 3 h after heat shock, aliquots of cell lysates were immunoprecipitated with anti-Flag monoclonal antibody (MAb) resin. Bound proteins were eluted with 0.1 M glycine-HCl (pH 3.5), separated by SDS-polyacrylamide gel electrophoresis, transferred onto a Hybond ECL membrane, and probed with the anti-Flag monoclonal antibody (D). To detect coprecipitating Epi-tagged proteins, the membrane was stripped and reprobed with the anti-Epi monoclonal antibody (B). Expression of the tagged proteins was confirmed by immunoblotting aliquots of the total-cell lysates with the anti-Flag (C) or anti-Epi (A) monoclonal antibody. The identities of visualized proteins are indicated on the right; antibodies used for Western blotting are shown on the left. Lanes 9 are indicated by an asterisk and show a positive control for coprecipitation: Flag-tagged Reaper and Epi-tagged OpIAP.