Anti α tubulin

In brief, the total cell lysates were prepared in high KCl lysis buffer (10 mM Tris-HCl, pH 8.0, 140 mM NaCl, 300 mM KCl, 1 mM EDTA, 0.5% Triton X-100 and 0.5% sodium deoxycholate) with complete protease inhibitor cocktail (Roche).The protein concentration was determined using BCA (bicinchoninic acid) protein assay kit (Beyotime Biotech, Jiangsu, China) and store at -20 °C. Thirty micrograms of protein were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membranes (Roche). The membranes were treated with 1% blocking solution in TBS for 2 h, the membranes were incubated with primary antibodies anti-GSTP1 (1:200; Bioss, Beijing, China), anti-α-tubulin (1:1000; AbMart, Shanghai, China), at 4 °C overnight. Then the membranes were washed and incubated with POD-labeled secondary antibodies (Roche). The immunolabeled proteins were detected by BM Chemiluminescence Western Blotting kit (Roche).

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).