FIG. 2.

Translation initiation at start sites internal to the mdm2 ORF is responsible for the production of N-terminally truncated MDM2 isoforms. (A) Saos-2 cells were transfected with the indicated YFP-tagged MDM2 constructs (5 μg). Following immunoprecipitation by a mixture of MDM2 antibodies 2A9 and 2A10, precipitated proteins from lysates of the transfected cells were analyzed by immunoblotting (IB) using monoclonal MDM2 antibody 4B11 as probe. The top panel shows gel locations of bands corresponding to endogenous as well as exogenous MDM2. Bands resulting from expression of YFP-tagged MDM2 constructs and which have been separated in a lower percentage gel to achieve better resolution are shown in the bottom panel. Dots in both panels indicate the positions of faster-migrating MDM2 isoforms in which the N-terminal domain of the protein is absent. (B) MDM2 proteins in Saos-2 cells transfected with indicated constructs were analyzed as described for panel A, except that MDM2 antibodies 4B11 as well as 3G5 were used for probing sequentially in immunoblotting. The 3G5 antibody, which requires a native MDM2 segment between amino acids 59 and 89, fails to detect mutated MDM2-YFP (lane 4 and lane 5, bottom panel), possibly because the target protein has a substituted amino acid at position 62.