Anti phosphorylated gsk 3β p gsk 3β

Cells were lysed in RIPA lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 1 mM EDTA, 0.1% sodium dodecyl sulfate, 1 mM sodium vanadate, 1 mM NaF, 1 mM phenylmethanesulfonyl fluoride, 0.1 mg/ml pepstatin, 0.1 mg/ml leupeptin, and 0.1 mg/ml aprotinin). The protein concentration was determined by using a bicinchoninic acid protein assay. Protein lysates (40 μg) were then resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto polyvinylidene difluoride membranes (PVDF; Bio-Rad, Hercules, CA, USA), and blotted with different primary antibodies [anti-IL-18; Abcam, Cambridge, UK; anti-GSK-3β, anti-phosphorylated GSK-3β (p-GSK-3β), anti-caspase-3, anti-cleaved caspase-3, anti-caspase-7, anti-cleaved caspase-7; all from Cell Signaling Technology, Boston, MA, USA] overnight at 4°C. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies and visualized with an ECL reagent (GE Healthcare, London, UK).