Fig. 2

Mutant MDM2 (1–115) fragments do not bind to p53 and react with two conformation-dependent antibodies in vitro

(A) Five different plasmids were translated and [³⁵S]-labeled in vitro: luciferase as a negative control, full length and 1–115 of human wild-type Mdm2 as positive controls, and the two mutant 1–115 fragments identified in the yeast screen. These translation products were then precipitated by incubation with GST-fusion proteins on glutathione beads and were separated on a 15% SDS-Polyacrylamide gel. CSB is a negative control fusion of amino acids 1–242 of CSB DNA repair ATPase to GST (GST::CSB (1–242)). p53 is a fusion of amino acids 1–82 of p53 to GST (GST::p53 (1–82)). p53 failed to precipitate the mutant MDM2 proteins. (B) The same in vitro translation products used in A were immunoprecipitated as indicated and were separated on a 15% SDS-Polyacrylamide gel. “Poly” is polyclonal sera against MDM2; 419 is a negative control monoclonal antibody against SV40 T antigen; 3G5 and 4B2 are MDM2-specific monoclonal antibodies that do not recognize denatured protein. Both mutant proteins were recognized by the conformation-dependent antibodies, which suggests that they are properly folded Molecular weight markers in kD are shown along left side.