Anti p gsk3β ser9 antibody

Cellular proteins were extracted using protein extraction reagent (Novagen, Madison, WI, USA) supplemented with a protease inhibitor cocktail (Calbiochem, San Diego, CA, USA) on ice. Protein concentration was determined with a protein assay kit (Bio-Rad, Hercules, CA, USA). Next, proteins (30 μg) were separated on a 10% SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride membrane (Bio-Rad). The membrane was incubated with specific primary antibodies overnight at 4 °C. Primary antibodies included anti-Nestin antibody (1:1000, Millipore), anti-MAP2 antibody (1:1000, Millipore), anti-GFAP antibody (1:1000, Millipore), anti-p-GSK3β (Ser9) antibody (1:1000, Cell Signaling, Beverly, MA, USA), anti-GSK3β antibody (1:1000, Cell Signaling), anti-Non-p (active) β-catenin antibody (1:1000, Cell Signaling), and anti-β-catenin antibody (1:1000, Cell Signaling). The membrane was then incubated with the secondary antibody for 1 h at room temperature. β-Actin antibodies (1:5000, Sigma) were used as the reference. The bands were visualized and imaged using the ChemiDoc Touch imaging system (Bio-Rad).