Protein a and g beads

IP and western blotting were performed as our previous report.^{24 (link)} Briefly, cells were transfected, treated with 10 μM MG132 for 6 h, and lysed using 2 × RIPA buffer (Tris-HCl, pH 7.4 (100 mM), NaCl (300 mM), 1% NP-40, 2% sodium deoxycholate, 10 mM NaF, and 10 mM Na vanadate). The cell lysates were cleared by centrifugation and incubated with 1 μg antibody for 1 h at 4 ^°C followed by incubation with 15 μl protein A and G beads (Santa Cruz, Santa Cruz, CA, USA) for 2 h at 4 ^°C. Immunoprecipitates were subjected to western blotting. For western blotting analysis, cells were scraped from the dishes into the lysis buffer. A total of 25 mg of total protein was separated by SDS-PAGE and blotted with anti-c-Myc (Epitomics, Burlingame, CA, USA), anti-NPM (Epitomics), anti-EBP2 (Abnova, Taipei City, Taiwan, China), anti-β-catenin (CST, Danvers, MA, USA), anti-IκB-α (CST), anti-α-tubulin (Abmart, Shanghai, China), anti-actin (Abmart), or anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Abmart).