Animals were sacrificed after 8 weeks on the diet regimen and perfused with PBS. Segments of the thoracic aorta and the entire skeletal muscle were immediately frozen in TissueTek® Optimal Cutting Temperature Compound on dry ice. Ten micrometer sections were rehydrated in PBS, permeabilized with 0.3 % Triton-X-100, and incubated overnight with rabbit anti-glucose transporter GLUT4 antibody (Abcam, Cambridge, MA, ab654) at 1:50 followed by Alexa Fluor®568 goat anti-rabbit IgG (Invitrogen) at 1:800. Stained sections were mounted in fluorescence mounting medium containing DAPI. Images were collected under controlled exposure and gain settings with an Eclipse 80i (Nikon, Tokyo, Japan) with SPOT™ software (SPOT™ Imaging Solutions, Sterling Heights, MI). Six aortic and 2 skeletal muscle sections from each of 3 WT and 4 KO mice per diet group were stained for GLUT4. The mean fluorescence intensity of GLUT4 staining in the skeletal muscle was determined using ImageJ to quantify the fluorescence intensity of 20 myofibers from each mouse.
Glut4 antibody
Manufactured by Abcam
Sourced in United States, United Kingdom
GLUT4 antibody is a protein-specific antibody that recognizes the GLUT4 (Glucose Transporter 4) protein, which is responsible for insulin-stimulated glucose uptake in adipose and muscle tissues. This antibody can be used for the detection and analysis of GLUT4 expression in various biological samples.
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Lab products found in correlation
Irs 1, by Cell Signaling Technology (3 mentions)
Trilobatin, by Merck Group (3 mentions)
Myod1, by Cell Signaling Technology (3 mentions)
Rat mouse insulin elisa kits ezrmi 13k, by Merck Group (3 mentions)
Ecl chemiluminescence detection reagent, by Merck Group (3 mentions)
Plasma membrane protein extraction kit, by Beyotime (3 mentions)
Nuclear cytosolic fractionation kit, by Beyotime (3 mentions)
β actin, by Cell Signaling Technology (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions)
Palmitate, by Merck Group (3 mentions)
Ripa buffer, by Beyotime (3 mentions)
Bca protein assay kit, by Beyotime (3 mentions)
Speci c anti mouse and anti rabbit hrp conjugated second antibodies, by Santa Cruz Biotechnology (2 mentions)
Attune nxt, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 568 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Eclipse 80i, by Nikon (1 mentions)
Specific anti mouse and anti rabbit hrp conjugated second antibodies, by Santa Cruz Biotechnology (1 mentions)
Flotillin 2, by Cell Signaling Technology (1 mentions)
Anti syntaxin 4 rabbit polyclonal antibody, by Synaptic Systems (1 mentions)
Zdhhc5 antibody, by Merck Group (1 mentions)
10 protocols using glut4 antibody
1
Quantifying GLUT4 Expression in Aortic and Skeletal Muscle Tissue
2
Glucose Uptake and Insulin Signaling Pathway
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Trilobatin, palmitate, DAPI and (2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino)-2-deoxyglucose (2NBDG) uptake measurement kits were purchased from Sigma (St. Louis, MO, USA). IRS1, p-IRS1 (Ser 612), p-IRS1 (Ser 307), Akt, p-Akt (Ser 473), p-Akt (thr308), Na, K-ATPase, MYH1, MYOD1, and β-actin primary antibodies were bought from Cell Signaling Technology (Danvers, MA, USA). GLUT4 antibody was obtained from Abcam (Cambridge, MA, USA). HRP-conjugated GAPDH primary antibody was purchased from Aksmics (shanghai, China), Specific anti-mouse and anti-rabbit HRP-conjugated second antibodies were obtained from Santa Cruz Biotechnology (Texas, CA, USA). Rat/mouse insulin ELISA kits (EZRMI-13K) and ECL chemiluminescence detection reagent were obtained from Millipore (Billerica, MA, USA). Plasma membrane protein extraction kit, nuclear/cytosolic fractionation kit, RIPA buffer and BCA protein assay kit and other chemicals were purchased from Beyotime (Shanghai, China).
3
Antibody sources for protein detection
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Monoclonal Flotillin-2 and monoclonal IRAP antibodies were obtained from Cell Signalling (MA, USA). GLUT4 antibody was from Abcam (Cambridge, UK). Anti-Syntaxin 4 rabbit polyclonal antibody was purchased from Synaptic Systems (Goettingen, Germany). zDHHC5 antibody was from Sigma (Poole, UK). Anti-HA rat monoclonal antibody was purchased from Roche (Basel, Switzerland).
4
Western Blot Analysis of GLUT4
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For western blot analysis, 30 μg protein was utilized. Blots were probed with specific primary antibodies and the appropriate secondary antibodies (Jackson ImmunoResearch Lab. West Grove, PA). β-actin was used as a loading control. GLUT4 antibody was purchased from Abcam, Cambridge, MA.
5
Glut4 Expression Quantification in Cells
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Treated E4 and NV cells were trypsinized and blocked in blocking buffer (PBS/3% FCS) for 30 min at 4°C. Following blocking, cells were centrifuged at 5000 rpm, 4°C, for 5 min, supernatant was aspirated and cells were incubated with Glut4 antibody (1:50) (Abcam #ab65267) in PBS/3% FBS for 1 h at 4°C, and washed 3X with PBS/3%FBS. Cells were stained with anti-mouse Alexa 647 antibody (Thermofisher Scientific #A31571, 1:50) at 4°C for 1 h, washed 3X with PBS/3% FBS, and fixed with 2% paraformaldehyde for 15 min at RT. Cells were analyzed with flow cytometer (Attune NxT, Thermofisher Scientific) with cytometer settings for both forward scatters (FSC) and side scatters (SSC) set to 80 and 600 respectively to analyze cells. For each sample, 5000 cells were counted.
6
Quantifying Protein Expression in Tissues
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Total protein was extracted from the muscle or liver tissues by homogenization in the radioimmunoprecipitation assay (RIPA) buffer (sigma). The homogenate was centrifuged at 14,000 rpm for 20 minutes and supernatant that contained the proteins was removed. Total protein was estimated by the Bradford method. Using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), the proteins were separated by loading 120 μg protein/lane. The proteins were then transferred onto a Polyvinylidene difluoride (PVDF) membrane. Blocking was performed by overnight incubation of the membrane in 5% non-fat skim milk in a Tris-Buffered Saline and Tween 20 (TBST) buffer solutions at 4° C.
The membrane was incubated with appropriate polyclonal primary antibodies (Abcam) (PPARγ or GLUT4 antibody) in a TBST buffer for one hour, and then washed thrice with a TBST buffer (20 minutes each), followed by incubation with anti-rabbit secondary antibody for one hour, at room temperature. Next, the PVDF membrane was incubated with a substrate (western lightening plus ECL, Perkin-Elmer) for one minute. Following this, in a dark room, the PVDF was exposed to Hyblot film (Denvill) for 30 seconds. After development, the band densities were analyzed by the ImageJ software.
The membrane was incubated with appropriate polyclonal primary antibodies (Abcam) (PPARγ or GLUT4 antibody) in a TBST buffer for one hour, and then washed thrice with a TBST buffer (20 minutes each), followed by incubation with anti-rabbit secondary antibody for one hour, at room temperature. Next, the PVDF membrane was incubated with a substrate (western lightening plus ECL, Perkin-Elmer) for one minute. Following this, in a dark room, the PVDF was exposed to Hyblot film (Denvill) for 30 seconds. After development, the band densities were analyzed by the ImageJ software.
7
Regulation of AMPK and MAPK Signaling
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Glucose, insulin, curcumin, and 1, 7-bis (4-hydroxy-3-methoxyphenyl)-1, 6-heptadiene-3, 5-dione (DHZ) were purchased from Sigma-Aldrich (St. Louis, MO, USA). 5-Aminoimidazole-4-carboxamide-1-β-ribofuranoside (AICAR) was purchased from Toronto Research Chemical Incorporation (Toronto, ON, Canada). DHZ and SB203580 [a p38 mitogen-activated protein kinase (MAPK) inhibitor] were obtained from BIOMOL International LP (Butler Pike, PA, USA). Polyclonal anti-phospho AMPKα, p38 MAPK, insulin receptor substrate-1 (IRS-1), Akt and inactive AMPKα, p38 MAPK, IRS-1, Akt and anti-β-actin antibodies were purchased from Millipore Millipore, (Billerica, MA, USA) (MA, USA). GLUT4 antibody was purchased from Abcam (Cambridge, UK). Compound C, an AMPK inhibitor, was provided by Merck (RY 70-100; Rahway, NJ, USA). Hybond ECL nitrocellulose membrane was obtained from Amersham (Arlington Heights, IL, USA). All cell culture reagents and other chemicals were purchased from Life Technologies (Gaithersburg, MD, USA).
8
Trilobatin Effects on GLUT4 Expression
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After the mice were administrated with 10 mg/kg trilobatin for 4 weeks (once for each day), the skeletal muscle tissues were isolated and fixed (10% formalin solution in 0.1 M PBS), frozen at − 80 °C overnight, and cut into 10 μm sections on a freezing microtome (Leica, Nussloch, Germany). The sections were permeabilized in 0.1% Triton X-100 in PBS and blocked in 1% BSA in PBS before incubation with the primary antibody. After stained with GLUT4 antibody (ab654, Abcam Inc. Cambridge, MA, USA) and together with DAPI nuclear stain (Invitrogen, CA, USA). Fluorescence images were acquired using a confocal microscope (Nikon, Tokio, Japan).
9
Insulin Signaling and Glucose Uptake Assay
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Trilobatin, palmitate, DAPI and (2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino)-2-deoxyglucose (2NBDG) uptake measurement kits were purchased from Sigma (St Louis, MO, USA). IRS1, p-IRS1 (Ser 612), p-IRS1 (Ser 307), Akt, p-Akt (Ser 473), p-Akt (thr308), Na,K-ATPase, MYH1, MYOD1, and β-actin primary antibodies were bought from Cell Signaling Technology (Danvers, MA, USA). GLUT4 antibody was obtained from Abcam (Cambridge, MA, USA). HRP-conjugated GAPDH primary antibody was purchased from Aksmics (shanghai, China), Speci c anti-mouse and anti-rabbit HRP-conjugated second antibodies were obtained from Santa Cruz Biotechnology (Texas, CA, USA). Rat/mouse insulin ELISA kits (EZRMI-13K) and ECL chemiluminescence detection reagent were obtained from Millipore (Billerica, MA, USA). Plasma membrane protein extraction kit, nuclear/cytosolic fractionation kit, RIPA buffer and BCA protein assay kit and other chemicals were purchased from Beyotime (Shanghai, China).
10
Glucose Uptake and Insulin Signaling
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Trilobatin, palmitate, DAPI and (2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino)-2-deoxyglucose (2NBDG) uptake measurement kits were purchased from Sigma (St Louis, MO, USA). IRS1, p-IRS1 (Ser 612), p-IRS1 (Ser 307), Akt, p-Akt (Ser 473), p-Akt (thr308), Na,K-ATPase, MYH1, MYOD1, and β-actin primary antibodies were bought from Cell Signaling Technology (Danvers, MA, USA). GLUT4 antibody was obtained from Abcam (Cambridge, MA, USA). HRP-conjugated GAPDH primary antibody was purchased from Aksmics (shanghai, China), Speci c anti-mouse and anti-rabbit HRP-conjugated second antibodies were obtained from Santa Cruz Biotechnology (Texas, CA, USA). Rat/mouse insulin ELISA kits (EZRMI-13K) and ECL chemiluminescence detection reagent were obtained from Millipore (Billerica, MA, USA).
Plasma membrane protein extraction kit, nuclear/cytosolic fractionation kit, RIPA buffer and BCA protein assay kit and other chemicals were purchased from Beyotime (Shanghai, China).
Plasma membrane protein extraction kit, nuclear/cytosolic fractionation kit, RIPA buffer and BCA protein assay kit and other chemicals were purchased from Beyotime (Shanghai, China).
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