Ge10600001

Colon tissues or colon tumors were extracted, frozen and pulverize using a liquid nitrogen-cooled mortar. The same amount of powder samples was lysed in buffer containing 1% NP40, 150 mM NaCl, 50 mM Tris HCl pH 7.5, 2 mM EDTA, 2 mM EGTA, 20 mM sodium fluoride, 2 mM PMSF, 2 mM sodium orthovanadate, 1 mM DTT and 1x EDTA-free complete protease inhibitor cocktail (Roche, #11873580001), and sonicated at high intensity for 5 min with Standard Bioruptor Diagenode. Protein content was quantified using DC Protein Assay (Biorad), and 30 μg of total protein lysate in Laemmli buffer was separated on SDS-PAGE and transferred to nitrocellulose membrane (GE10600001, Sigma). After blocking (5% non-fat milk and 1% BSA in PBS, 1hat room temperature) membranes were incubated at 4°C overnight with anti-CPEB4 (Abcam) or anti-CPEB4 monoclonal (149C/D10, homemade) primary antibodies or at room temperature for 1 h with anti-vinculin (ab130007, AbCam) and anti-b-Actin (ab8224, AbCam) primary antibodies. After incubation with secondary antibodies for 1hat room temperature, the membranes were incubated with Amersham ECL™ Western Blotting Detection Reagents (GERPN2106, Sigma).