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Institute of Metabolism and Endocrinology, Department of Clinical Laboratory, Department of Orthopaedics, The Second Xiang-Ya Hospital of Central South University, 410011 ChangSha, People's Republic of China
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's Republic of China). In addition, the PCR products were extracted from the agarose gels and sequenced by BioAsia Company. Cell treatment and western blotting analysis To detect the phosphorylation of AKT, the direct target in IRS1 pathway, after the IRS1
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blocking for 60 min at room temperature in Tris-buffered saline containing 0.1% Tween 20 and 5% nonfat milk, the membranes were incubated overnight at 4°C with anti-pAkt (Ser473) antibody (Cell Signaling, #9271), anti-Akt antibody (Cell Signaling, #9272
Shanghai Key Laboratory of Endocrine Tumor, Division of Endocrinology and Metabolism, Shanghai Jiao-Tong University School of Medicine, Shanghai Institute of Endocrinology and Metabolism, Shanghai Clinical Center for Endocrine and Metabolic Diseases, Rui-Jin Hospital, 197 Rui-Jin 2nd Road, Shanghai 200025, People's Republic of China
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Shanghai Key Laboratory of Endocrine Tumor, Division of Endocrinology and Metabolism, Shanghai Jiao-Tong University School of Medicine, Shanghai Institute of Endocrinology and Metabolism, Shanghai Clinical Center for Endocrine and Metabolic Diseases, Rui-Jin Hospital, 197 Rui-Jin 2nd Road, Shanghai 200025, People's Republic of China
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Shanghai Key Laboratory of Endocrine Tumor, Division of Endocrinology and Metabolism, Shanghai Jiao-Tong University School of Medicine, Shanghai Institute of Endocrinology and Metabolism, Shanghai Clinical Center for Endocrine and Metabolic Diseases, Rui-Jin Hospital, 197 Rui-Jin 2nd Road, Shanghai 200025, People's Republic of China
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Shanghai Key Laboratory of Endocrine Tumor, Division of Endocrinology and Metabolism, Shanghai Jiao-Tong University School of Medicine, Shanghai Institute of Endocrinology and Metabolism, Shanghai Clinical Center for Endocrine and Metabolic Diseases, Rui-Jin Hospital, 197 Rui-Jin 2nd Road, Shanghai 200025, People's Republic of China
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Shanghai Key Laboratory of Endocrine Tumor, Division of Endocrinology and Metabolism, Shanghai Jiao-Tong University School of Medicine, Shanghai Institute of Endocrinology and Metabolism, Shanghai Clinical Center for Endocrine and Metabolic Diseases, Rui-Jin Hospital, 197 Rui-Jin 2nd Road, Shanghai 200025, People's Republic of China
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Shanghai Key Laboratory of Endocrine Tumor, Division of Endocrinology and Metabolism, Shanghai Jiao-Tong University School of Medicine, Shanghai Institute of Endocrinology and Metabolism, Shanghai Clinical Center for Endocrine and Metabolic Diseases, Rui-Jin Hospital, 197 Rui-Jin 2nd Road, Shanghai 200025, People's Republic of China
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Shanghai Key Laboratory of Endocrine Tumor, Division of Endocrinology and Metabolism, Shanghai Jiao-Tong University School of Medicine, Shanghai Institute of Endocrinology and Metabolism, Shanghai Clinical Center for Endocrine and Metabolic Diseases, Rui-Jin Hospital, 197 Rui-Jin 2nd Road, Shanghai 200025, People's Republic of China
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metabolism, can be activated in peripheral tissues by two distinct pathways. One is insulin signaling pathway via PI3-K/Akt. Insulin accelerates glucose transport via the phosphorylation and activation of Akt, leading to glucose transporter 4 (GLUT4
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polyclonal), anti-JAK2 (sc-278, rabbit polyclonal), anti-Akt (sc-1618, goat polyclonal), anti-phospho [Thr 183 /Tyr 185 ]JNK (sc-6254, mouse monoclonal), anti-phosphotyrosine (pY) (sc-508, mouse monoclonal), anti-phospho Ser 473 Akt (sc-7985-R, rabbit
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expression or activity. Broken arrow: indirect effect. P indicates phosphorylation. ACT2BR, activin 2B receptor; AMPK, AMP-activated protein kinase; AS160, Akt substrate of 160 kDa; CRFR2, corticotrophin-releasing factor receptor 2; FOXO, forkhead
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-AMPK (Cell Signaling), anti-phospho-ACC and anti-ACC (Cell Signaling), anti-phospho-Akt and anti-Akt (Cell Signaling) and anti-β-actin antibodies (Santa Cruz). The level of each protein was quantified using the ImageJ program and normalized to the level of β
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), phospho-Akt (Ser 473 ), Akt, Akt substrate of 160 kDa (AS160), phospho-AS160 (Thr 642 ), c-Jun NH 2 -terminal kinase (SAPK/JNK), phospho-SAPK/JNK (Thr 183 /Tyr 185 ), p38 mitogen-activated protein kinase (MAPK) and phospho-p38 MAPK (Thr 180 /Tyr 182
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Physiology, Departments of
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H5% medium (without TSH) for 7 days were treated with or without TSH (1 mU/ml), 30 μM LY294002 (LY294), 30 μM LY303511 (LY303), 3 μM PI-103, 30 μM of a casein kinase II inhibitor (CK2i), and/or 30 μM of an Akt1/Akt2 selective inhibitor (Akti-1
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) containing 5% nonfat dry milk for 1 h and incubated with the appropriate antibodies overnight. The following primary antibodies were used: rabbit anti-Akt1/2 (H-136) Sc-8312 (1:1000; Santa Cruz Biotechnology), rabbit anti-p-Akt1/2/3 (Ser473) Sc-7985-R (1
Departments of, Human Nutritional Sciences, Physiology, Molecular Physiology Laboratory, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2
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Departments of, Human Nutritional Sciences, Physiology, Molecular Physiology Laboratory, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2
Departments of, Human Nutritional Sciences, Physiology, Molecular Physiology Laboratory, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2
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Departments of, Human Nutritional Sciences, Physiology, Molecular Physiology Laboratory, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2
Departments of, Human Nutritional Sciences, Physiology, Molecular Physiology Laboratory, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2
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). Following electrophoresis, the proteins were transferred to PVDF membrane and probed with primary antibodies to eukaryotic elongation factor 2 kinase (eEF2K), phospho-eEF2K, phosphorylated extracellular-regulated kinase (ERK1/2), phospho-Akt, phospho