Figure 5
ELISA-based in vitro MDM2/p53 protein-protein interaction assay.
(A), (B) Different amount of cell lysates (panel A) or human recombinant MDM2/p53 protein (panel B) were captured on wells pre-coated with MDM2 antibody. After extensive washes, levels of the MDM2/p53 complex were quantified using an antibody specific for p53 and subsequently an HRP-conjugated antibody and a TMB substrate kit. (C), (D) U87MG cell lysates, containing the native MDM2/p53 complex, were pre-incubated with DMSO (control) or different concentrations of the indicated compounds. Then, lysates were captured on wells pre-coated with MDM2 antibody. After extensive washes, levels of the MDM2/p53 complex were quantified using an antibody specific for p53 and subsequently an HRP-conjugated antibody and a TMB substrate kit. Blank wells were obtained in the absence of p53 antibody. Data are expressed as absorbance at 450 nm minus blank values (A), (B), or as % of control set to 100% (C), (D) and represent the mean ± SEM of at least three independent experiments.
