Rabbit anti human gsk 3β

Cells were washed with cold phosphate buffered saline (PBS) and lysed in Pro-Prep solution (Intron, Korea) containing a phosphatase inhibitor cocktail (Sigma, Saint Louis, MO). After lysis, protein quantification was performed using Bradford assays (Biorad, Hercules, CA). Equal amounts of protein were resolved in sodium dodecyl sulfate (SDS)-polyacrylamide gels. Separated proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Biorad, Hercules, CA). Subsequently, membranes were incubated for 30 minutes in blocking solution (5% nonfat milk), and incubated overnight at 4°C with mouse anti-human IL-32α (Biolegend, San Diego, CA), mouse anti-human α-tubulin (Sigma, Saint Louis, MO), rabbit anti-human β-catenin, rabbit anti-huamn phospho-β-catenin, rabbit anti-human GSK-3β, rabbit anti-human E-cadherin, rabbit anti-human phospho-p44/42 MAPK or rabbit anti-human total p44/42 MAPK (Cell Signaling, Danvers, MA) antibodies (1:1000). After incubation with the primary antibody, membranes were washed three times with PBS containing 0.1% Tween 20 (Merck, Germany) and incubated for 1 hour at room temperature with horseradish peroxidase (HRP)-conjugated secondary antibodies (Jackson Laboratory, West Grove, PA). Target proteins were visualized using an ECL system (Amersham Biosciences, UK) and LAS3000 (Fuji Film).