25-OCH₃-PPD destabilizes MDM2 protein.

<p>(A) MCF7 and MDA-MB-468 cells were treated with DMSO or 25-OCH₃-PPD (25 µM) followed by exposure to protein synthesis inhibitor cycloheximide (CHX, 10 µg/mL). MDM2 levels were detected by Western blotting at indicated times after exposure to CHX. (B) MCF7 cells were transfected with MDM2 and ubiquitin plasmids followed by treatment with various concentrations of 25-OCH₃-PPD for 24 h. Cells were harvested (left panel) or exposed to proteasome inhibitor MG132 (25 μM) for additional 6 h (right panel). Ubiquitinated MDM2 was detected by immunoblotting. (C) The cell lysates of MCF7 cells treated as above were immunoprecipitated with anti-MDM2 antibody. The ubiquitinated MDM2 was detected using anti-ubiquitin antibody.