Mdm2 smp 14

5×10⁶ EU-1 cells in 3 mL RPMI-1640 culture media (supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin) were seeded in a 6 well-plate and incubated for 12 h, then 1 mL of the drug-loaded medium was added. The cells were harvested at a designated time point, washed with cold PBS then centrifuged at 1500 rpm (3 times). The cells were lysed by adding 200 μL of cold NP-40 buffer (supplied with cOmplete™ protease Inhibitor tablet (Roche) and 1mM PMSF). The cell lysates were centrifuged at 12500 rpm at 4 °C for 10 min and total protein concentration in the supernatant was measured with the BCA assay (Thermo-Fisher). 30 μL of the cell lysate was mixed with 10 μL 4x laemmli sample buffer and denatured at 95°C for 5min. The total protein concentration was adjusted with lx laemmli sample buffer. Equal amount of protein sample was loaded onto 4-15% gradient SDS PAGE gel (Bio-rad). After electrophoresis, the protein was transfer to PTFE membrane (Bio-rad) and probed with the corresponding antibody. MDM2 ((SMP14), 1:500 Santa Cruz); p53 ((DO-1), 1:800, Santa Cruz); GAPDH ((0411), 1:2000, Santa Cruz) and HRP conjugated goat anti-mouse secondary antibody (Bio-rad, 1:2000). The chemiluminescent result was detected with Pierce ECL Plus Substrate (Thermo Scientific) and imaged with LSA4000 (GE Healthcare, Fairfield, USA).