Western blot was performed as previously described.16 , 17 , 18 Cytoplasmic and nuclear protein fractions were extracted from cells using NE‐PER Nuclear and Cytoplasmic Extraction Reagents (Pierce, Rockford, IL, USA) according to the manufacturer's protocol. The primary antibodies included PIK3CD (Santa Cruz Biotechnology, Santa Cruz, CA, USA, 1:1000), AKT (Cell Signaling Technology, Beverly, MA, USA, 1:1000), phosphorylated (phospho)‐AKT (Ser473) (Cell Signaling Technology, 1:500), glycogen synthase kinase (GSK)‐3β (Cell Signaling Technology, 1:1000), phospho‐GSK‐3β (Ser9) (Cell Signaling Technology, 1:1000), β‐catenin (Cell Signaling Technology, 1:1000), phospho‐β‐catenin (Ser33/37/Thr41) (Cell Signaling Technology, 1:1000), C‐myc (Abcam, Cambridge, MA, USA, 1:1000), CyclinD1 (Abcam, 1:5000), GAPDH (Santa Cruz, 1:2000) and Histone 3 (Cell Signaling Technology, 1:2000). GAPDH or Histone 3 was used as a loading control.
Glycogen synthase kinase 3β
Manufactured by Cell Signaling Technology
Sourced in United States
Glycogen synthase kinase (GSK)-3β is a serine/threonine protein kinase that plays a key role in the regulation of glycogen synthesis, cell cycle, and cell survival. It is involved in the phosphorylation of glycogen synthase, which inhibits its activity, thereby regulating glycogen synthesis.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Cell Signaling Technology (3 mentions)
Phosphorylated pkbser473, by Cell Signaling Technology (2 mentions)
Phosphorylated gsk 3βser9, by Cell Signaling Technology (2 mentions)
Pik3cd, by Santa Cruz Biotechnology (1 mentions)
Phospho β catenin ser33 37 thr41, by Cell Signaling Technology (1 mentions)
Histone 3, by Cell Signaling Technology (1 mentions)
Phospho gsk 3β ser9, by Cell Signaling Technology (1 mentions)
Cyclin d1, by Abcam (1 mentions)
C myc, by Abcam (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
β catenin, by Cell Signaling Technology (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
β catenin, by Abcam (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Wnt3a, by Abcam (1 mentions)
Bca protein assay kit, by Wuhan Boster Biological Technology (1 mentions)
Radioimmunoprecipitation assay lysis buffer, by Wuhan Boster Biological Technology (1 mentions)
Phosphorylated crebser133, by Cell Signaling Technology (1 mentions)
Protein assay kit, by Bio-Rad (1 mentions)
Imagequant tl, by Cytiva (1 mentions)
5 protocols using glycogen synthase kinase 3β
1
Western Blot Analysis of Signaling Pathways
2
Western Blot Analysis of MSCs Signaling Pathways
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The MSCs from three normal healthy subjects were randomly selected and seeded into 6-well plates at 1×105 cells/well after four culture passages. Total protein was extracted using radioimmunoprecipitation assay lysis buffer (Wuhan Boster Biological Technology, Ltd.). Protein concentration was quantified using a BCA Protein Assay kit (Wuhan Boster Biological Technology, Ltd.). The protein samples (20 µg) were separated by 10% SDS-PAGE and transferred onto a polyvinylidene difluoride membrane (EMD Millipore). Following blocking with 5% skimmed milk powder at 37°C for 1 h, the membranes were incubated with primary antibodies against Smad9 (cat. no. ab115900; 1:500; Abcam), Wnt3a (cat. no. ab28472; 1:500; Abcam), β-catenin (cat. no. 8480; 1:1,000; Cell Signaling Technology, Inc.), glycogen synthase kinase (GSK)-3β (cat. no. 12456; 1:1,000; Cell Signaling Technology, Inc.) and β-actin (cat. no. 4970; 1:1,000; Cell Signaling Technology, Inc.) at 4°C overnight, followed by a HRP-conjugated goat anti-rabbit immunoglobulin G secondary antibody (cat. no. BA1054; 1:5,000; Wuhan Boster Biological Technology, Ltd.) at room temperature for 1 h. The protein bands were visualized using a ChemiDoc™ MP imaging system (Bio-Rad Laboratories, Inc.). Protein levels were calculated relative to β-actin.
3
Hippocampal Protein Expression Analysis
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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 CREBser133, protein kinase B (PKB or Akt), phosphorylated PKBSer473, glycogen synthase kinase (GSK)-3β, phosphorylated GSK-3βser9, tau, phophorylated tauser396 and β-actin (Cell Signaling Technology). The intensity of protein expression was determined using Imagequant TL (Amersham Biosciences).
4
Reagents for Metabolic Signaling Assays
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Roswell Park Memorial Institute (RPMI)-1640 medium were purchased from Gibco. Fetal bovine serum (FBS) was acquired from Lonsera. Insulin was purchased from Novo Nordisk. Adenyl cyclase activators, forskolin (Fsk) and dexamethasone (Dex), were obtained from Beyotime Biotech Inc. The AMPK inhibitor and activator (Compound C) and A-769662 were purchased from Med Chem Express. Primary antibodies against AKT, phospho-Ser473 AKT, glycogen synthase kinase (GSK)-3β, phospho-GSK-3β, AMPK, phospho-Thr172 AMPK, phospho-ACC, and α-tubulin were obtained from Cell Signaling Technology. Thermo Fisher Scientific was used to obtain a rabbit polyclonal antibody against ACOT4. Other primary antibodies against phosphoenolpyruvate carboxylase (PEPCK) and peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) were obtained from Proteintech Group, Inc. Glucose-6-phosphatase (G6pase) antibody was acquired from Abclonal. Anti-FLAG tag antibody was acquired from Sigma-Aldrich.
5
Differentiated PC12 cell signaling
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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 PKBSer473, glycogen synthase kinase (GSK) 3β, phosphorylated GSK-3βser9, 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).
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