Western Blot Analysis

Prepare protein extracts as previously described.

Homogenize tissues in lysis buffer (0.33Molarity (M) sucrose/8millimolar (mM) Hepes, 7.4 and protease inhibitors) and quantify them using the BCA protein determination method (Bio-Rad, Hercules, CA).

Dilute protein samples to equivalent volumes containing 20µg of protein and boil in an equal volume of Laemli SDS boiling buffer (Sigma) for 0h 10m 0s.

Load samples into a 9-12% SDS-polyacrilamide gel and separate it by electrophoresis for 3h 0m 0s at 100 V.

Transfer the proteins to polyvinylidene difluoride membrane (Amersham Biosciences, Piscataway, NJ) for 1h 30m 0s at 300 mA.

After blocking of nonspecific binding with 5% non-fat dry milk in TBST, probe the membranes with primary antibodies and process.

Perform densitometric analysis using ImageQuantity One.

Normalize data to β-actin, normalize values of phosphorylated GSK-3β (pTyr²¹⁶ GSK-3β); phosphorylated α-syn (pSer¹²⁹ α-syn) and phosphorylated tau (pSer³⁹⁶ tau) to total GSK-3β, α-syn, and tau, respectively, before statistical analysis of variance and express values as percent changes (%) of WT controls.

Dashed lines (in white) indicate discontinuous bands (nonsequential lanes) taken from the same blot, at the same molecular weight (mass – kDa) in order to better represent the mean signal from all values (5-6 individual blots/genotype/treatment) for that particular group. Corresponding control bands (loading controls) match experimental bands.