Phosphorylated gsk 3β s9

XAV939 was obtained from Selleckchem (Houston, TX, USA). PF4800567 was purchased from Sigma Chemical (St. Louis, MO, USA). Anti-PP2A, Anti-CK1εand anti-Dvl2 antibodies were obtained from GeneTex (Irvine, CA, USA). Phosphorylated Dvl2 (S143) antibody were purchased from Abcam (Cambridge, MA, USA). Phosphorylated β-catenin (S33, S37 and T41), GSK-3β, and phosphorylated GSK-3β (S9) antibodies were purchased from Cell Signaling (Danvers, MA, USA). All other antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

SH-SY5Y and murine brain homogenates were analyzed for various proteins through Western blotting. Proteins were separated by SDS-PAGE with 10 μg per lane for SH-SY5Y homogenates and 15μg for murine homogenates. Protein was transferred to PVDF membrane and blocked with 5% BSA. Primary (dilutions below) and secondary antibodies (1:2500 dilution) were incubated on blots in 0.5% BSA. Antibodies used in this study are:
AT8 [phosphorylated S202/T205 tau] (Thermo) 1:500 mouse IgG, AT180 [phosphorylated T231/S235 tau] (Thermo) 1:500 mouse IgG, PHF-1 [phosphorylated S396/S404 tau] (Peter Davies) 1:500 mouse IgG, pT231 [phosphorylated T231 tau] (Thermo) 1:1000 rabbit IgG, Tau12 (Skip Binder) 1:5000 mouse IgG, GSK-3β total (Cell Signal) 1:1000 mouse IgG, Phosphorylated-GSK-3β S9 (Cell Signal) 1:1000 rabbit IgG, CDK5 (Cell Signal) 1:1000 rabbit IgG, p35/p25 (Cell Signal) 1:500 rabbit IgG, and GAPDH (Thermo) 1:5000 mouse IgG. Blots were imaged using a Bio-Rad Gel Doc XR Imaging platform with Image Lab software. Images were exported as *.tiff, quantified using Image J, and assembled using Adobe Illustrator.