GST Pulldown Assay.

H1299 transiently transfected with MDMXc3 was lysed in lysis buffer [50 mM Tris⋅HCl (pH 8.0), 5 mM EDTA, 150 mM NaCl, 0.5% Nonidet P-40, 1× protease inhibitor mixture] and digested with PreScission. Bacterial lysate expressing GST-p53 fusion proteins were applied to glutathione-agarose beads according to the manufacturer instruction (Pierce). The beads (20 µL of packed volume) loaded with ∼1 µg of GST fusion proteins were incubated for 2 h at 4 °C with PreScission-digested H1299 lysate. The beads were washed with lysis buffer, boiled in Laemmli sample buffer, and detected by Western blot for MDMX fragments.