Fig. 7.

PEPD blocks MDM2-mediated p53 move to mitochondria. a IB analysis of MDM2 in cells treated with siRNA for 72 h. b IB analysis of p53 in cells treated with siRNA and 24 h later treated with or without MG132 (25 μM) for 48 h. c IB and IP-IB analyses of p53 association with MDM2 after cell treatment with siRNA for 48 h. d IP-IB analysis of MDM2 binding to the p53-PEPD complex. e IB analysis of mitochondria translocation of p53 in cells treated first with control siRNA or MDM2 siRNA and 24 h later treated with control siRNA or PEPD siRNA for 48 h. MDM2 siRNA #1 was used (see Methods). f IB analysis of mitochondrial monoubiquitinated p53 in cells treated with siRNA for 48 h, with UbAl (100 μM) added in the final 4 h. GAPDH, α-tubulin and VDAC were measured to ensure the purity of subcellular fractions or as loading controls. Cells were cultured in 6-well plates (5–10 × 10⁴ cells/well) for 24 h before experimental treatment