Figure 6.

MDM2 monoubiquitinates DHFR and reduces DHFR activity in a RING finger–dependent manner. A, an in vitro ubiquitination assay was performed using GST-MDM2 or GST as indicated. The panels on the right are from a separate experiment. Indicated amounts of dialyzed DHFR were incubated with 200 ng GST-MDM2 or GST control protein in the presence or absence of ubiquitin. Proteins were resolved by SDS-PAGE, Western blotted, and probed with an anti-DHFR antibody. B, brilliant blue G colloidal stained SDS-PAGE gel indicating the amounts of GST-MDM2 or GST protein used in the ubiquitination reactions. C, H1299 cells were transfected with the indicated plasmids, and cell lysates were assayed for DHFR activity. Three independent transfections were performed for each condition. The right hand panel shows immunoblot analysis of the indicated proteins from the same lysates. D, as for C but with a RING finger mutant of MDM2 included, and transfection was performed with varying amounts of each plasmid DNA as indicated. Note the cutoff of the Y axis in C. Three independent transfections were performed for each condition and for each weight of plasmid DNA used for transfection. Columns, mean; bars, SE. Statistical analysis by Student’s t test. Each experiment has been repeated on at least three occasions, and the results shown are from a single typical experiment.