Rabbit anti gsk 3β

Cultivated cells were harvested and lysed using lysis buffer [1% (v/v) Triton X-100, 200 mM HEPES (pH 7.9), 300 mM NaCl, 100 mM KCl, 10 mM EDTA, 10 µg/ml aprotinin, 100 µg/ml leupeptin, and 10 µM PMSF]. Twenty micrograms of total protein lysate was electrophoresed in an acrylamide gel and transferred to a TransBlot^® membrane (162–0145, Bio-Rad, Hercules, CA, USA). Membranes were blocked in 5% (w/v) milk, incubated with primary antibodies for 1 h at room temperature, washed and incubated for 1 h with the appropriate HRP-conjugated secondary antibodies (sc-2004 and sc2005, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). The blots were washed in PBS containing 0.05% (v/v) Tween-20^® (P1379, Sigma-Aldrich Korea) and an ECL kit (34080, Pierce Biotechnology, Rockford, IL, USA) was used prior to detection of protein bands. The antibodies used were: rabbit anti-prohibitin (sc-28259, Santa Cruz Biotechnology, Inc.), rabbit anti-β-catenin (sc-7199, Santa Cruz Biotechnology, Inc.), rabbit anti-GSK-3β (sc-9166, Santa Cruz Biotechnology, Inc.), rabbit anti-p-GSK-3β (sc-11757-R, Santa Cruz Biotechnology, Inc.), mouse anti-c-Myc (sc-40, Santa Cruz Biotechnology, Inc.), and mouse anti-β-actin monoclonal (sc-8432, Santa Cruz Biotechnology, Inc.) to determine equal loading.