Phosphorylated gsk 3βser9

The day after hyperglycemic clamp, four overnight-fasted rats were randomly selected from 10 rats of each group and the hippocampi from the four rats were isolated. They were lysed in 20 mM Tris buffer (pH 7.4) containing 2 mM EDTA, 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 lysates using a Bio-Rad protein assay kit, lysates with equal amounts of protein were immunoprecipitated with specific antibodies prior to separation by SDS-PAGE as previously described [23 (link),30 (link)]. Antibodies used for the immunoblot analysis were cAMP responding element binding protein (CREB), phosphorylated CREB^ser133, protein kinase B (PKB or Akt), phosphorylated PKB^Ser473, glycogen synthase kinase (GSK)-3β, phosphorylated GSK-3β^ser9, tau, phophorylated tau^ser396 and β-actin (Cell Signaling Technology). The intensity of protein expression was determined using Imagequant TL (Amersham Biosciences).

The methods were described as previously described [3 (link)]. Anti-GSK-3β (#9832), AKT (pan) (#4685), phosphorylated AKT (Ser473) (#4060), GSK-3β (Ser9) (#12456) and phosphorylated GSK-3β (Ser9) (#5558) were purchased from Cell Signaling Technology. Anti-CDK5RAP3 (ab157203), E-cadherin (ab1416), N-cadherin (ab76057), Vimentin (ab8978), and GAPDH (ab181602) antibodies were purchased from Abcam. AKT siRNA (#6211) and control siRNA (#6568) were purchased from Cell Signaling Technology. We corrected the loading error based on loading controls and compared the expression levels of target proteins in gastric tumor and adjacent non-tumor tissues. The protein expression in tumors was defined as high level when it was higher than that in normal tissue but was defined as low level when it was lower than that in normal tissue.

Western blotting was performed to determine the expression levels of phosphorylated GSK3β (ser9; 9336, Cell Signaling Technology, Beverly, MA, USA), total GSK3β (9315, Cell Signaling Technology), phosphorylated GSK3α (8542, Cell Signaling Technology), total GSK3α (4818, Cell Signaling Technology), total β-catenin (9582, Cell Signaling Technology), myogenin (F5D, Developmental Studies Hybridoma Bank, Iowa City, IA, USA) and pan-myosin heavy chain (MHC) (MF20, Developmental Studies Hybridoma Bank). Solubilized proteins from tissue homogenates and cell lysates were separated using 4–15% TGX BioRad precast gels and then transferred onto 0.2 μm polyvinylidene difluoride (PVDF) membranes. Subsequently, membranes were blocked with 3% bovine serum albumin in tris-buffered saline tween solution (TBS-T) and then immunoprobed with their corresponding primary antibodies. Membranes were then washed in TBS-T and immunoprobed with horseradish peroxidase-conjugated secondary antibodies. Chemiluminescent substrates, Immobilon (Millipore, Burlington, MA, USA), or Clarity Max (Bio-Rad, Hercules, CA, USA) were used to detect antigen–antibody complexes and were visualized using a BioRad ChemiDoc imager. Quantification of optical densities was performed using ImageLab (BioRad, USA) and normalized to total protein visualized using Ponceau staining.

Differentiated PC12 cells with NGF were treated with the vehicle, BA730-E, BA730-W, BA731-E, and BA731-W for 24 h and the cells were administered with 2 nM insulin for 30 min. The cells were harvested with lysate buffer and immunoblot analysis was conducted. Antibodies used for the immunoblot analysis were protein kinase B (PKB or Akt), phosphorylated PKB^Ser473, glycogen synthase kinase (GSK) 3β, phosphorylated GSK-3β^ser9, and β-actin (Cell Signaling Technology, Danvers, MA, USA). The intensity of protein expression was analyzed by Imagequant TL (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA).