Fig. 3. Mdm2 interacts with PML in vitro. (A) PML isoforms III, IV, IV-3K and PML-RAR were translated in vitro and mixed with equal amounts of in vitro translated wild-type Mdm2. The translation products were precipitated with an Mdm2 antibody mix (IF2, 2A10, SMP14) and blotted against PML (H-238) and Mdm2. Recognition of the different PML forms by the H-238 antibody was verified in separate experiments. (B) Mdm2 C-terminus is required for PML interaction. Mdm2 deletion mutants were translated in vitro and precipitated with in vitro translated PML IV. The products were precipitated with the Mdm2 antibody mix and blotted against the indicated proteins. Deletion constructs 58-89 and 89-222 were analysed in separate experiments together with full-length Mdm2. Note that Mdm2 migrates as three bands because of alternative translation initiation. As a negative control, in vitro translated Mdm2 was omitted from the binding reaction (neg. ctrl).