Image studio software v 2

Hippocampi were dissected from hemibrains over ice and homogenized in ice‐cold Pierce RIPA buffer containing Complete protease inhibitor cocktail and phosphatase inhibitor cocktails 2 and 3. The homogenates were sonicated twice for 5 min at 40 A using an EpiSonic Multi‐Functional Bioprocessor (Epigentek, Farmingdale, NY, USA). Proteins (20 μg/well) were separated on a 4–12% NuPAGE Bis‐Tris gel (Life Technologies, South San Francisco, CA, USA) and transferred onto nitrocellulose membranes for 9 min using iBlot (Life Technologies). After blocking for 1 h in a solution of 5% bovine serum albumin in Tris‐buffered saline (BSA‐TBS), membranes were incubated overnight at either room temperature with an antibody against phosphorylated SYN (1:500, Wako, Richmond, VA, USA) or at 4°C with antibodies against SYN (1:1000, Millipore) or α‐tubulin (1:50,000, Sigma‐Aldrich, St. Louis, MO, USA). Antibodies were diluted in BSA‐TBS containing 0.1% Tween 20 (BSA‐TBST). All membranes were incubated for 1 h at room temperature in a solution of IRDye 800CW‐tagged or 680LT‐tagged donkey anti‐rabbit or donkey anti‐mouse secondary antibodies (1:10,000, Li‐Cor, Lincoln, Nebraska, USA) in Odyssey blocking buffer with 0.2% Tween 20. Signals were detected with an Odyssey CLx laser scanner (Li‐Cor) and quantified using Image Studio software v2.1.10 (Li‐Cor).