In vitro characterization of RPS15 mutations. (A-B) RPS15^G132A and RPS15^P131S interact with MDM2 and MDMX. Immunoblot analysis of whole-cell lysates (input) and immunoprecipitates (IP) of HCT116 cells cotransfected with 1 of the Myc-DDK-RPS15 vectors (wt, G132A or P131S) and either tGFP-MDM2 (A) or tGFP-MDMX (B). Twenty-four hours posttransfection 500 μg of cell lysates were subjected to immunoprecipitation using anti-tGFP antibody followed by immunoblotting with indicated antibodies. Immunoglobulin G heavy chain was used as loading control for IP fraction. (C-D) Transient expression of RPS15^wt, RPS15^P131S, and RPS15^G132A in HCT116 cells revealed an impaired ability to stabilize endogenous p53, in particular for the RPS15^G132A mutant. Western blot images from 1 representative experiment and quantification results from 3 independent experiments are shown. Images from 2 additional experiments are presented in supplemental Figure 7. (E) Ubiquitination experiments revealed increased ubiquitin-mediated p53 degradation in both RPS15 mutants (40.1% and 40.6% increase for RPS15^P131S and RPS15^G132A, respectively, measuring the intensity for the upper 4 bands) compared with wild-type RPS15.