Gsk 1β

The liver collected from rats stimulated with insulin for 10 min was lysed with lysis buffer containing a 20 mM Tris buffer (pH 7.4) containing 2 mM EGTA, 137 mM NaCl, 1% NP40, 10% glycerol, and 12 mM α-glycerol phosphate and protease inhibitors. After 30 min on ice, the lysates were centrifuged for 10 min at 12,000 rpm at 4°C. After measuring protein contents in lysate by Bio Rad protein assay kit (Hercules, CA), lysates with equivalent amounts of protein (30–50 μg) were resolved with SDS-PAGE and immunoblotted with antibodies of phospho-Akt^ser478, Akt, phospho-glycogen synthase kinase- (GSK-) 1β, GSK-1β (Cell Signaling Technology, Beverly, MA), and phosphoenolpyruvate carboxykinase (PEPCK), generously provided by Dr. Garner of Vanderbilt University [16 (link), 19 (link)]. The intensity of protein expression was determined using ImageQuant TL (Amersham Biosciences, Piscataway, NJ). These experiments were repeated three times for each group.

Hypothalami collected from six rats stimulated with insulin for 10 min were lysed with RIPA lysis buffer containing protease inhibitors. After measuring protein contents in the lysate using the Bio-Rad protein assay kit (Hercules, CA, USA), lysates with equivalent amounts of protein (30–50 μg) were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotted with antibodies to phosphorylated Akt^ser478, Akt, phosphorylated glycogen synthase kinase (GSK)-1β, and GSK-1β (Cell Signaling Technology, Beverly, MA, USA), [16 (link),18 (link)]. The intensity of protein expression was determined using Imagequant TL (Amersham Biosciences, Piscataway, NJ, USA). These experiments were repeated three times for each group.