Miz 1

Whole cell extracts were prepared by resuspending cells in RIPA buffer (50 mM Tris-HCl pH 8, 150 mM NaCl, 1% NP-40 substitute, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with complete protease inhibitor cocktail (Roche). Cleared lysates were boiled in Laemmli sample buffer with 1% beta-mercaptoethanol and proteins were separated by SDS-PAGE using precast 4–12% Bis-Tris gels (Life Technologies). Proteins were transferred to PVDF membrane (Millipore) at 30 V, 4 °C for 16 h. Blots were blocked for 30 min with 5% milk in TBS-T, incubated with primary antibodies for >3h and horseradish peroxidase conjugated anti-mouse IgG or anti-rabbit IgG (GE Healthcare) for 1 h, washing 4× for 10 min with excess TBS-T in between and prior to developing. Blots were developed with SuperSignal West Pico ECL substrate (Thermo Scientific) and exposed to film. The following antibodies were used in this study: Huwe1 (Bethyl, A300–486A, 1:1000), N-myc (Santa Cruz, B8.4.B, 1:500), c-Myc (Cell Signaling, 9402, 1:500), Mcl-1 (Santa Cruz, S-19, 1:200), Miz-1 (Santa Cruz, H-190, 1:200), beta catenin (Cell Signaling, 8480, 1:1000), Erk1/2 (Cell Signaling, 4695, 1:1000), phospo-Erk1/2 (Cell Signaling, D13.14.4E, 1:1000), p53 (Leica, CM5, 1:1000), vinculin (Sigma, hVIN-1, 1:5000) and actin (EMD Millipore, C4, 1:5000).