The following antibodies were used: skeletal muscle myosin (Santa Cruz Biotechnology, Dallas, TE, USA), Glut4 (Proteintech, Rosemont, IL, USA), Myomesin1 (Proteintech), Hexokinase II (Cell Signaling Technology), FoxO1 (Cell Signaling Technology), phospho-FoxO1 (Cell Signaling Technology) (rabbit), Histone H3 (Cell Signaling Technology), Glut1 (Cell Signaling Technology), GAPDH (Cell Signaling Technology), Vinculin (Cell Signaling Technology), Desmin (Milipore/Sigma-Aldrich), Troponin-I (Milipore/Sigma-Aldrich), b-Actin (mouse; Milipore/Sigma-Aldrich), anti-mouse HRP (GE Healthcare, Chicago, IL, USA), and anti-rabbit HRP (GE Healthcare). AS1842856 was purchased from Adooq.
Glut4
Manufactured by Proteintech
Sourced in United States
GLUT4 is a protein that functions as a glucose transporter. It is responsible for facilitating the uptake of glucose into cells, particularly in insulin-responsive tissues such as skeletal muscle and adipose tissue.
Automatically generated - may contain errors
Lab products found in correlation
Pgc 1α, by Proteintech (2 mentions)
Gapdh, by Proteintech (2 mentions)
Histone h3, by Cell Signaling Technology (1 mentions)
Glut1, by Cell Signaling Technology (1 mentions)
Phospho foxo1, by Cell Signaling Technology (1 mentions)
Vinculin, by Cell Signaling Technology (1 mentions)
As1842856, by Adooq (1 mentions)
Hexokinase 2, by Cell Signaling Technology (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Foxo1, by Cell Signaling Technology (1 mentions)
Collagen 1, by Abcam (1 mentions)
Connexin 43, by Thermo Fisher Scientific (1 mentions)
Collagen 3, by Abcam (1 mentions)
Total ampk, by Cell Signaling Technology (1 mentions)
Nf κb, by Proteintech (1 mentions)
Tgf β, by Abcam (1 mentions)
β actin, by Proteintech (1 mentions)
Cpt1b, by Proteintech (1 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Protein extraction kit, by Beyotime (1 mentions)
8 protocols using glut4
1
Molecular Profiling of Skeletal Muscle
2
Western Blot Analysis of Protein Levels
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Protein levels were measured by WB with β-actin as the loading control. Protein was extracted from atria samples or cells and quantitated by a BCA assay. Equal amount (10–30 μg) of protein was loaded and separated by SDS-PAGE using 10 or 12% acrylamide gradients, and later transferred to nitrocellulose membranes. The membranes were blocked with 5% skim milk and incubated with antibodies against total-AMPK (1:1,000; Cell Signaling Technology/CST, Massachusetts, United States), CD36 (1:1,000; Abcam), collagen Ⅰ (1:1,000; Abcam), collagen Ⅲ (1:1,000; Abcam), connexin-43 (1:200; Invitrogen), CPT1B (1:1,000; Proteintech), GLUT4 (1:500; Proteintech), NFκB (1:1,000; Proteintech), NRF2 (1:1,000; Proteintech), phoso-AMPK (Thr172; 1:1,000; CST), phoso-Akt (Ser473; 1:1,000; CST), phoso-NFκB (1:1,000; CST), PGC1α (1:1,000; Proteintech), SOD2 (1:1,000; Proteintech), TGF-β (1:1,000; Abcam), α-SMA (1:1,000; CST), β-actin (1:5,000; Proteintech). Protein levels were quantified as the intensity of bands using ImageJ software (NIH systems) and standardized to β-actin. Abbreviations are fully illustrated in corresponding figure legends.
3
Comprehensive Metabolic Protein Analysis
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The BCA protein content detection kit was bought from KeyGen Biotech (Nanjing, China). Hypersensitive ECL chemiluminescence kit, protease, and phosphatase inhibitor were bought from NCM Biotech (Suzhou, China). The protein extraction kit was obtained from Beyotime Institute of Biotechnology (Shanghai, China). Non-esterified fatty acids (NEFA) and blood lipid test kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Revert Aid First Strand cDNA Synthesis Kit was purchased from Thermo Fisher Scientific (Waltham, MA, United States). Power SYBR Green PCR master mix was from Invitrogen (Carlsbad, CA, United States). The primers of AMPKα, SIRT1, PGC-1α, PPARα, GLUT4, UCP3, and β-actin were designed and synthesized by Sangon Biotech (Shanghai, China). Both primary antibodies to AMPKα, PGC-1α, PPARα, GLUT4, UCP3, GAPDH, Lamin B1, and Na, K-ATPase (Cat no: 66536-1-Ig, 66369-1-Ig, 15540-1-AP, 66846-1-Ig, 10750-1-AP, 60004-1-Ig, 66095-1-Ig, and 14418-1-AP) and secondary antibodies (Cat no: SA00001-1 and SA00001-2) were purchased from the Proteintech Group (Chicago, IL, United States). The antibody to Phospho-AMPKα (Thr172, Cat #: 50081S) was purchased from the Cell Signaling Technology (Boston, MA, United States). The antibody to SIRT1 (Cat #: ab189494) was purchased from Abcam (Cambridge, United Kingdom).
4
Adipocyte Protein Extraction and Analysis
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Total proteins from 3T3-L1 adipocytes were isolated by radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, Haimen, China) containing phosphatase inhibitor (Beyotime, Haimen) and protease inhibitor cocktail (Biosharp). The membrane protein was extracted by using a membrane and cytosol protein extraction kit (Beyotime, Haimen). The protein concentration was determined by the BCA protein assay kit (Beyotime, Haimen). The proteins were separated by 8% or 12% sodium dodecyl sulfate-polyacrylamide gel and then transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 5% nonfat dry milk for 1 h and incubated with different primary antibodies: NF-kB p65, p-NF-kB p65 (Ser536), IL-6, TNF-α (Affinity, Cambridge, United Kingdom), PI3K, p-PI3K (Tyr458/Tyr199), AKT, p-AKT (Ser473) (Cell Signaling Technology, Beverly, MA, United States), and GLUT4 (Proteintech, Chicago, IL, United States) at 4°C overnight. After washing with Tris-buffered saline containing Tween 20 (TBST), the membrane was incubated with the specific secondary antibody for 1 h at room temperature with shaking. Finally, the membrane surface was uniformly coated with the ECL chemiluminescent substrate reagent (Biosharp) and then exposed to the Image Lab analysis system (Bio-Rad, ChemiDocXRS+, United States). The protein expression was normalized to the tubulin level.
5
Protein Analysis Workflow with SDS-PAGE
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The protein concentration was quantified with the BCA Protein Assay Kit. Then 4× protein gel electrophoresis loading buffer was added to the protein samples, which were denatured at 100°C for 5 min. The SDS-PAGE gel was prepared, samples were added for electrophoresis, and electricity was transferred to PVDF membrane. Seal with 5% skim milk powder sealing solution for 2 h, adding primary antibody and incubating overnight at 4°C: Tubulin (1:10000, GTX628802) and Caspase-3(1:1000, #14220) from CST; p-AMPK (1:2000, AP0116) and HO-1(1:1000, A1346) from ABclonal; PI3K (1:1000, AF6241) from Affinity; LKB1 (1:1000, 10746-1-AP), p-LKB1 (1:5000, 80127-1-RR), AMPK (1:500, 10929-2-AP), p-AKT (1:5000, 66444-1-Ig), AKT (1:5000, 60203-2-Ig), GLUT4 (1:3000, 66846-1-Ig) from Proteintech; Bax (1:1000, GB12690) from Seville; Bcl-2 (1:1000, BA0412) and Nrf2 (1:1000, A0674) from BOSTER; Lamin-B1(1:1000, SI17-06) from Huabio. After washing, the membrane was incubated for 1 h with second antibody (1:8000, Servicebio, China) and the proteins were detected with ECL luminescent solution. The gray value was analyzed by Image J software.
6
Western Blot Analysis of Insulin Signaling
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7
Adipogenesis Regulatory Protein Analysis
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The antibodies recognizing PPARγ, PPARα, C/EBPα, Glut4, LPL, FAS, and aP2 were purchased from Proteintech (Wuhan, China); the antibodies recognizing β-actin and ACC1 were purchased from BOSTER (Wuhan, China); the antibody recognizing SREBP1 was purchased from Bioss (Beijing, China); and the antibody recognizingSCD1 was purchased from Abcam (United Kingdom).
8
Adipose Tissue Molecular Profiling
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Each iWAT and eWAT was collected at the end of the experiment, and western blot analysis was performed as previously described. The primary antibodies included: anti β-actin (Proteintech), anti-AMPKα (Cell Signaling Technology), anti-p-AMPKα (Cell Signaling Technology), SIRT1 (Abcam), anti-AKT (Proteintech), anti-p-AKT (Proteintech), GLUT4 (Proteintech), IRS1 (Proteintech) and PPARγ (Abcam).
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