Mouse anti akt

Mice were decapitated rapidly at 30 min after i.p. injection of leptin. The hippocampi were dissected out and homogenized in lysis buffer (50 mM HEPES pH 7.6, 1% triton X-100, 150 mM NaCl, 20 mM sodium pyrophosphate, 20 mM b-glycerophosphate, 10 mM NaF) containing a mixture of phosphatase inhibitors (leupeptin, aprotinin, sodium orthovanadate, phenylmethylsulfonyl fluoride, Ser/Thr phosphatase inhibitor mixture, Tyr phosphatase inhibitor mixture). A total amount of 40 μg protein was separated by SDS-PAGE, and transferred to a nitrocellulose membrane. The membrane was blocked in a blocking buffer (0.01 M Tris-buffered saline, 0.01M Tris base, 0.9% NaCl with 1% dry milk and 0.1% Tween 20) followed by incubation with mouse anti-Akt (1:1000; Invitrogen, Carlsbad, CA) and rabbit anti-pAkt^Thr308, pAkt^Ser473 or Akt antibodies (1: 1000, Cell Signaling Technology Inc., Danvers, MA) diluted in a solution of 1% bovine serum albumin and 0.1% Tween-20 in Tris-buffered saline overnight at 4°C. Horseradish peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulin G was used as secondary antibodies (1:5000, Cell Signaling). Signals were detected by enhanced chemilluminescence (Thermo Scientific, Rockford, IL). Quantification of Western blotting was performed using ImageJ software with normalization to total protein levels.