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Glut2

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Glut2 is a glucose transporter protein that facilitates the movement of glucose across cell membranes. It plays a key role in glucose homeostasis and is primarily expressed in the liver, pancreas, and intestine.

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Lab products found in correlation

β actin, by Merck Group (2 mentions) Acc1 2, by Cell Signaling Technology (1 mentions) Pampkt172, by Cell Signaling Technology (1 mentions) Oxphos cocktail, by Abcam (1 mentions) Glut4, by Santa Cruz Biotechnology (1 mentions) Pakt1 s473, by Cell Signaling Technology (1 mentions) Cleaved caspase 3, by Cell Signaling Technology (1 mentions) Bromodeoxyuridine (brdu), by Abcam (1 mentions) Insulin, by Abcam (1 mentions) Caspase 3, by Cell Signaling Technology (1 mentions) Glucagon, by Agilent Technologies (1 mentions) Tead1, by Abcam (1 mentions) Lsm 780, by Zeiss (1 mentions) Lsm 510, by Zeiss (1 mentions) Cryostat, by Leica (1 mentions) E cadherin, by Merck Group (1 mentions) Rabbit gfp antibody, by Thermo Fisher Scientific (1 mentions) Somatostatin, by Agilent Technologies (1 mentions) Pancreatic polypeptide, by Agilent Technologies (1 mentions) Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

Anti-glut2 (11 citations) GLUT2 (07-1402) and Pex14 (ABC-142) (2 citations) Rabbit anti-Glut2 (2 citations) Rabbit anti‐GLUT2 (Polyclonal; (2 citations)

6 protocols using glut2

1

Western Blot Analysis of Metabolic Proteins

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Tissues designated for western blot were harvested and snap frozen in liquid nitrogen. Tissues were lysed and homogenized as described [26 ]. Modifications to the protocol include a substitution to a 2X RIPA lysis buffer and the addition of two extra centrifugation steps at 10,000g for 5 minutes to reduce the congealed lipid levels found in the liver lysates. Protein concentrations were then quantified with Coomassie Bradford Assay. 20–50 μg of samples were electrophoresed at 4° C and transferred overnight (16 hours, 20V at 4°C). Primary antibodies used are pAMPKT172 (1:2000; Cell Signaling Technology), AMPK (1:2000; Cell Signaling Technology), Glut4 (1:750; Santa Cruz Biotechnology), Glut2 (1:2000, Millipore), HKII (1: 10,000; Millipore), phospho-Glycogen SynthaseS641/S645 (pGS) (1:10,000; Invitrogen), pACC1/2 (1:8,000; Cell Signaling Technology), ACC1/2 (1:8,000; Cell Signaling Technology), OxPhos cocktail (1:2000; AbCam), VDAC (1:7,000; AbCam), pAKT1S473 (1:2000;Cell Signaling Technology), AKT1 (1:2000;Cell Signaling Technology). Bands at predicted sizes were acquired on a ChemiDoc ™ XRS Systems. Optical densities were quantified using Image Lab software and normalized against total protein content acquired through Ponceau S staining or β-actin (1:3000, Sigma Life Science).
Heydemann A., González-Vega M., Berhanu T.K., Mull A.J., Sharma R, & Holley-Cuthrell J. (2016). Hepatic Adaptations to a High Fat Diet in the MRL Mouse Strain are Associated with an Inefficient Oxidative Phosphorylation System. Jacobs journal of diabetes and endocrinology, 2(1), 013.
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2

Pancreatic Immunostaining and Morphometric Analysis

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Mouse pancreas were harvested, embedded in paraffin and sectioned to 5 µm thickness.
Immunostaining was performed as previously described (Lee et al., 2013) . Primary antibodies used were Glut2 (rabbit polyclonal, 1:200; Millipore), Insulin (guinea pig ployclonal, 1:500; Abcam), Glucagon (rabbit polyclonal, 1:200; Dako), Tead1 (rabbit polyclonal, 1:200; Abcam), Taz (rabbit polyclonal, 1:200; Santa Curz), Yap (rabbit poly clonal 1:200; Cell signaling), p16 (mouse monoclonal, 1:1,000; Abcam), Pdx1 (guinea pig polyclonal, 1:500; Abcam), Mafa (rabbit polyclonal, 1:100; Bethyl lab), Ucn3 (rabbit polyclonal, 1:2,000; MGI), Ki67 (rabbit poly clonalm 1:50; Abcam), Brdu (rat polyclonal, 1:1,000; Abcam), Caspase-3 (rabbit polyclonal, 1:200; Cell signaling), and Cleaved Caspase-3 (rabbit monoclonal, 1:200; Cell signaling). To measure total pancreatic islet area, pancreas paraffin blocks from mice were cut 5 µm thickness sections spaced 100 µm apart between each slide. 5 pancreatic sections per mouse were stained and measured islets area using ImageJ software. For assessing number of Brdu positive and Ki67 expression cells in β-cell, 3,000 to 5,000 cells were counted from mouse pancreatic sections in 3 to 4 mice from each group.
Lee J., Liu R., Kim B.S., Zhang Y., Li F., Jagannathan R., Yang P., Negi V., Danvers J., Perez-Garcia E.M., Saha P., Sabek O.M., Creighton C.J., Coarfa C., Huising M.O., Shih H., Bottino R., Ma K., Moulik M., & Yechoor V. (2020). Tead1 reciprocally regulates adult β-cell proliferation and function to maintain glucose homeostasis.
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3

Immunofluorescence Analysis of Pancreatic Cell Markers

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Pancreas tissues were fixed overnight in 4% paraformaldehyde, incubated in 30% sucrose, and then embedded in O.T.C. medium (Tissue-Tek, Hatfield, PA). Frozen sections (10 microns) were prepared using a Leica cryostat (Leica) and then incubated with primary antibodies against insulin, E-cadherin (Sigma, St. Louis, MO), glucagon, somatostatin, pancreatic polypeptide (Dako, Carpinteria, CA), Glut2 (EMD Millipore, WA), Pdx1 (Abcam, Cambridge, MA), Isl-1, Sox9, Hnf6, ghrelin (Santa Cruz, CA), Ptf1a (Beta Cell Biology Consortium), Nkx2.2, Nkx6.1, Ngn3 (Developmental Studies Hybridoma Bank), Amylase (Cell Signaling Technology, Danvers, MA), and GFP (rabbit GFP antibody, Life Technologies; chicken GFP antibody, Abcam), with the FITC-conjugated lectin Dolichos biflorus agglutinin (DBA), a ductal cell marker or DAPI. After washing, the sections were subsequently incubated with anti-mouse, anti-chicken, anti-rabbit or anti-rat Alexa Fluor-488, Fluor-594, or Fluor-647 conjugated secondary antibodies (Life Technologies, Grand Island, NY). Fluorescence was observed in a Zeiss LSM780 or Zeiss LSM510 confocal microscope. Pancreata from at least 3 mice and at least 5 different sections from each pancreas were analyzed.
Kang H.S., Takeda Y., Jeon K, & Jetten A.M. (2016). The Spatiotemporal Pattern of Glis3 Expression Indicates a Regulatory Function in Bipotent and Endocrine Progenitors during Early Pancreatic Development and in Beta, PP and Ductal Cells. PLoS ONE, 11(6), e0157138.
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4

Western Blot Analysis of Glucose Transporters

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Using our previously published method, proteins were extracted and concentrations determined [16 (link)]. Next, 20 μg of protein were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes. After blocking nonspecific binding sites for 2 hours in TBST (1X TBS, 0.1% Tween 20) containing 4% non-fat milk and 1% bovine serum albumin (BSA), the membranes were incubated overnight on ice with the primary antibodies against SGLT1 (EMD Millipore Corporation, Billerica, MA, USA; 1:3000), GLUT2 (EMD Millipore Corporation; 1:3000), GLUT5 (EMD Millipore Corporation; 1:3000) and β-actin (Sigma-Aldrich; 1:3000). After washing three times in TBST, the membranes were incubated for 1 hour with horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies, and then visualized with enhanced chemiluminescence detection kit (Biological Industries, Beit Haemek, Israel) and intensities were quantified using Image-Pro plus 6.0 software (Media Cybernetics, Bethesda, MD, USA).
Li Z., Sun F., Zhang Y., Chen H., He N., Chen H., Song P., Wang Y., Yan S, & Zheng S. (2015). Tacrolimus Induces Insulin Resistance and Increases the Glucose Absorption in the Jejunum: A Potential Mechanism of the Diabetogenic Effects. PLoS ONE, 10(11), e0143405.
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5

Immunoblotting Analysis of Islet Proteins

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Human islets were prepared for experiments as described above, and mouse islets were isolated by collagenase digestion of whole mouse pancreas as previously described (27 ). Islets were transferred into fresh Miami 1A medium and treated with compounds at the concentration indicated in the figure legends for 24 hours at 37°C and 5% CO2. After the treatments, the islets were separated from the medium by centrifugation and frozen immediately in liquid nitrogen and maintained at –80°C until immunoblotting. Islet protein lysates were resolved by SDS-PAGE, transferred to nitrocellulose membranes (Bio- Rad), and blotted with primary Abs against Irs2 (EMD Millipore; no. MABS15); Irs1 mAb (produced in our laboratory); Pdx1 (Cell Signaling Technology; no. 5679S); FOXO1 (Cell Signaling Technology; no. 2880); FOXO3A (Cell Signaling Technology; no. 2497); CREB (Cell Signaling Technology, no. 9104); phosphorylated CREB (Cell Signaling Technology; no. 8212S); Glut2 (EMD Millipore; no. 07-1402); and tubulin (Cell Signaling Technology; no. 3873S). The density of bands was acquired and analyzed using a Kodak Imaging System and CareStream (version 5.0.7.23). Expression levels were normalized to tubulin as an internal standard.
Kuznetsova A., Yu Y., Hollister-Lock J., Opare-Addo L., Rozzo A., Sadagurski M., Norquay L., Reed J.E., El Khattabi I., Bonner-Weir S., Weir G.C., Sharma A, & White M.F. (2016). Trimeprazine increases IRS2 in human islets and promotes pancreatic β cell growth and function in mice. JCI Insight, 1(3), e80749.
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6

Protein Expression Profiling in Pancreas

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Western blot was performed as described by others (Zibolka et al. 2020) . Total protein from pancreas samples were extracted using RIPA lysis and extraction buffer (Thermo Fisher Scientific) supplemented with protease and phosphatase inhibitor cocktail (Roche, Basel, Switzerland). Equal amounts of protein (30 µg) were loaded to SDS-PAGE and transferred to nitrocellulose membrane. After blocking with 1% ROTI®Block (Carl Roth, Karlsruhe, Germany)
or 5% dry milk in TBS/T, blots were incubated with primary antibodies against insulin (1:1000, STJ24210; St John's Laboratory, London, United Kingdom), glucagon (1:500, PA5-13442;
Thermo Fisher Scientific), somatostatin (1:2000, STJ95730; St John's Laboratory), GLUT2
(1:4000, 07-1402, Merck Millipore, Burlington, USA) and GAPDH (1:1000, 3686, Cell Signaling Technology, Boston, USA). Followed by an incubation with horseradish peroxidase-conjugated secondary antibody, immunoreactions were detected by visualizing the peroxidase activity with an ECL Kit (Pierce™ ECL Western Blotting Substrate, Thermo Fisher Scientific).
For reloading the membrane with primary antibody, blots were stripped with western blot stripping buffer (Thermo Fisher Scientific) according to the manufacturer's instruction.
Brinker G., Froeba J., Arndt L., Braune J., Hobusch C., Lindhorst A., Bechmann I, & Gericke M. (2021). CD4+ T cells regulate glucose homeostasis independent of adipose tissue dysfunction in mice. European journal of immunology, 51(6).
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