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Glut3

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Glut3 is a laboratory equipment product that functions as a glucose transporter. It facilitates the movement of glucose across cell membranes.

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Lab products found in correlation

Vinculin, by Merck Group (3 mentions) Glut1, by Thermo Fisher Scientific (3 mentions) Slc7a11, by Cell Signaling Technology (3 mentions) Myc tag antibody, by Cell Signaling Technology (2 mentions) Protease and phosphatase inhibitor cocktail, by Roche (1 mentions) Protein a g bead, by Santa Cruz Biotechnology (1 mentions) Enhanced chemiluminescence system, by Merck Group (1 mentions) Phospho yaps127, by Cell Signaling Technology (1 mentions) Ampkα, by Santa Cruz Biotechnology (1 mentions) Tlr1 9 ligands, by InvivoGen (1 mentions) Hif 1α, by Santa Cruz Biotechnology (1 mentions) Eclipse te2000 e, by Nikon (1 mentions) 3 3 diaminobenzidine substrate, by Vector Laboratories (1 mentions) Nestin, by Thermo Fisher Scientific (1 mentions) βiii tubulin, by Merck Group (1 mentions) Glial fibrillary acidic protein (gfap), by Cell Signaling Technology (1 mentions) Eclipse c1 confocal microscope, by Nikon (1 mentions) Biotin conjugated anti rabbit igg, by Vector Laboratories (1 mentions) Cd133, by Miltenyi Biotec (1 mentions) Hrp conjugated secondary anti mouse or rabbit antibodies, by Bio-Rad (1 mentions)

Close spelling variants of this product

Anti-GLUT3 (4 citations) Rabbit anti-GLUT3 (3 citations) Mouse anti-GLUT3 (2 citations) GLUT3 (sc-30107) (2 citations)

10 protocols using glut3

1

Quantification of Metabolic Enzymes via Western Blot

Cited 2 times
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Western blotting was conducted as previously described 49 , 50 (link). Tumor tissues were powdered in a liquid nitrogen-cooled mortar and pestle and then lysed in 10 volumes of NP40 buffer as previously described 51 (link)–53 (link). The primary antibodies and concentrations used for Western blotting were following: G6PD (1:1,000, Cell Signaling Technology, #12263S), Vinculin (1:5,000, Sigma, #V4505), Myc tag antibody(1:2,000, Cell Signaling Technology, #2276S), TKT (1:1,000, Cell Signaling Technology, #8616S), PGD (1:1,000, Cell Signaling Technology, #13389S), SLC7A11 (1:2,000, Cell Signaling Technology, #12691S), GLUT1 (1:1,000, Thermo fisher, #PA5–16793), GLUT3 (1:1,000, santa cruz, #sc-74497), NRF2 (1:1,000, Cell Signaling Technology, # 12721S).
Liu X., Olszewski K., Zhang Y., Lim E.W., Shi J., Zhang X., Zhang J., Lee H., Koppula P., Lei G., Zhuang L., You M.J., Fang B., Li W., Metallo C.M., Poyurovsky M.V, & Gan B. (2020). Cystine transporter regulation of pentose phosphate pathway dependency and disulfide stress exposes a targetable metabolic vulnerability in cancer. Nature cell biology, 22(4), 476-486.
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2

Quantification of Metabolic Enzymes via Western Blot

Cited 2 times
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Western blotting was conducted as previously described 49 , 50 (link). Tumor tissues were powdered in a liquid nitrogen-cooled mortar and pestle and then lysed in 10 volumes of NP40 buffer as previously described 51 (link)–53 (link). The primary antibodies and concentrations used for Western blotting were following: G6PD (1:1,000, Cell Signaling Technology, #12263S), Vinculin (1:5,000, Sigma, #V4505), Myc tag antibody(1:2,000, Cell Signaling Technology, #2276S), TKT (1:1,000, Cell Signaling Technology, #8616S), PGD (1:1,000, Cell Signaling Technology, #13389S), SLC7A11 (1:2,000, Cell Signaling Technology, #12691S), GLUT1 (1:1,000, Thermo fisher, #PA5–16793), GLUT3 (1:1,000, santa cruz, #sc-74497), NRF2 (1:1,000, Cell Signaling Technology, # 12721S).
Liu X., Olszewski K., Zhang Y., Lim E.W., Shi J., Zhang X., Zhang J., Lee H., Koppula P., Lei G., Zhuang L., You M.J., Fang B., Li W., Metallo C.M., Poyurovsky M.V, & Gan B. (2020). Cystine transporter regulation of pentose phosphate pathway dependency and disulfide stress exposes a targetable metabolic vulnerability in cancer. Nature cell biology, 22(4), 476-486.
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3

Western Blot Analysis of Signaling Proteins

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Cells were lysed in ice-cold radioimmunoprecipitation assay (RIPA) buffer supplemented with a protease and phosphatase inhibitor cocktail (Roche). Whole-cell extracts were incubated with the indicated antibodies and protein A/G beads (Santa Cruz Biotechnology) overnight. The beads were washed with RIPA buffer three times and then boiled in SDS loading buffer. For Western blotting, cell extracts were separated by SDS-PAGE. After Western transfer, membranes were probed with antibodies against DDK (Origene ta50011), GFP (Santa Cruz biotechnology sc-101536), Glut3 (Santa Cruz biotechnology sc-74399), HA (Santa Cruz biotechnology sc-393579), PKM2 (Cell Signaling #3198), phospho-PKM2(Y705) (Cell Signaling #3827), YAP (Cell Signaling #12395), phospho-YAP(S127) (Cell Signaling #13008), actin (GeneTex GTX11003) and lamin C (GeneTex GTX101127). Bands were detected with an enhanced chemiluminescence system (Millipore). Western blotting was performed at least three times, and representative experiments are shown. Quantification was carried out using ImageJ software.
Kuo C.C., Ling H.H., Chiang M.C., Chung C.H., Lee W.Y., Chu C.Y., Wu Y.C., Chen C.H., Lai Y.W., Tsai I.L., Cheng C.H, & Lin C.W. (2019). Metastatic Colorectal Cancer Rewrites Metabolic Program Through a Glut3-YAP-dependent Signaling Circuit. Theranostics, 9(9), 2526-2540.
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4

Leukocyte Isolation and Endotoxin Response

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Leukocytes were isolated, lysed, and analyzed as described 18 (link). The antibodies used in Western blotting included LC3 and actin antibodies from Sigma (St. Louis, MI), and AMPKα, HIF-1α, and Glut3 antibodies from Santa Cruz Biotech (Santa Cruz, CA). ATP levels were determined using the ATP Bioluminescence Assay Kit HSII (Roch) as described 18 (link). Whole blood samples were incubated with endotoxin derived from Escherichia coliO111:B4 (Sigma; L3012) at 10 ng/ml for the indicated time. In other studies, whole blood samples were incubated with TLR 1-9 ligands (InVivoGen; San Diego, CA) for 2 hours. The leukocytes were then isolated and analyzed as described 18 (link).
Haimovich B., Zhang Z., Calvano J.E., Calvano S.E., Kumar A., Macor M.A., Corbett S., Coyle S.M, & Lowry S.F. (2014). Cellular metabolic regulators: Novel indicators of low-grade inflammation in humans. Annals of surgery, 259(5), 999-1006.
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5

Immunohistochemical Analysis of Neural Markers

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For immunohistochemical staining of formalin-fixed paraffin-embedded tissues, antigen retrieval was performed in citrate buffer at pH 6.0 and 95°C for 20 minutes. Sections were blocked then incubated overnight at 4°C in primary antibody integrin αvβ3 (LM609) or β3 (Cell signaling), Glut3 (Santa Cruz Biotechnology), GFAP (Cell Signaling), βIII tubulin (Sigma-Aldrich), Nestin (Fisher Scientific), CD133 (Miltenyi Biotech) followed by biotin-conjugated anti-rabbit IgG and an avidin-biotin peroxidase detection system with 3,3′-diaminobenzidine substrate (Vector Labs) and counterstained with hematoxylin. Double-immunostaining for β3/Glut3 was carried out according to manufacturer recommendations (Vector Labs). A Nikon Eclipse C1 Confocal microscope as well as a Nikon Eclipse TE2000-E were used for imaging.
Cosset É., Ilmjärv S., Dutoit V., Elliott K., von Schalscha T., Camargo M.F., Reiss A., Moroishi T., Seguin L., Gomez G., Moo J.S., Preynat-Seauve O., Krause K.H., Chneiweiss H., Sarkaria J.N., Guan K.L., Dietrich P.Y., Weis S.M., Mischel P.S, & Cheresh D.A. (2017). Glut3 addiction is a druggable vulnerability for a molecularly defined subpopulation of glioblastoma. Cancer cell, 32(6), 856-868.e5.
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6

Protein Extraction and Immunoblotting

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Proteins were extracted in RIPA buffer and quantified using the Pierce BCA kit (Thermo Fisher). 10–30μg of protein was boiled in NuPage buffer (Thermo Fisher) and loaded onto a denaturing SDS-polyacrylamide gel (10%), transferred to PVDF membranes and blotted with anti-mouse or -rabbit HRP-conjugated secondary antibodies (Bio-Rad). The following antibodies were used for immunoblotting: β3 (Cell Signaling), Glut3 (Santa Cruz Biotechnology), YAP (Santa Cruz), YAP-XP (Cell Signaling), TAZ (Cell Signaling), PAK4 and pPAK4 (Cell Signaling), and Vinculin and β-actin (Sigma-Aldrich) as loading controls.
Cosset É., Ilmjärv S., Dutoit V., Elliott K., von Schalscha T., Camargo M.F., Reiss A., Moroishi T., Seguin L., Gomez G., Moo J.S., Preynat-Seauve O., Krause K.H., Chneiweiss H., Sarkaria J.N., Guan K.L., Dietrich P.Y., Weis S.M., Mischel P.S, & Cheresh D.A. (2017). Glut3 addiction is a druggable vulnerability for a molecularly defined subpopulation of glioblastoma. Cancer cell, 32(6), 856-868.e5.
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7

Prefrontal Cortex Immunofluorescent Analysis

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The prefrontal cortices of mice were analyzed via immunofluorescent assays. Coronal sections (5-μm thickness) were prepared and immersed in 4% PFA for 30 min, followed by three washes in TBST. After blocking non-specific binding via 1.5% BSA for 1 h, sections were then incubated with corresponding primary antibodies overnight at 4 °C, as follows: GFAP (1:50; Proteintech, Wuhan, Hubei, China) and P-ERK1/2 (1:100; Beyotime, Shanghai, China); GFAP and GLUT3 (1:100; Santa Cruz, Dallas, Texas, USA); NeuN (1:50; Proteintech, Wuhan, Hubei, China); P-ERK1/2; and GFAP and T-ERK1/2 (1:100; Santa Cruz, Dallas, Texas, USA). After washing three times with TBST, the secondary antibody mix with FITC-conjugated goat anti-mouse IgG (1:200; Bioss, Beijing, China) and Cy3-conjugated goat anti-rabbit IgG (1:200; Bioss, Beijing, China) was added to cover the tissue and was incubated at room temperature for 1 h in the dark. Sections were then incubated with DAPI at room temperature for 5 min. Fluorescent images were obtained, and image analysis was applied to quantify immunoreactive signals.
Ouyang X., Wang Z., Luo M., Wang M., Liu X., Chen J., Feng J., Jia J, & Wang X. (2021). Ketamine ameliorates depressive-like behaviors in mice through increasing glucose uptake regulated by the ERK/GLUT3 signaling pathway. Scientific Reports, 11, 18181.
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8

Western Blotting of Tumor Tissue Lysates

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Western blotting was conducted as previously described (Liu and Gan, 2016 (link); Liu et al., 2016 (link)). Tumor tissues were ground to powder in a liquid nitrogen-cooled mortar and pestle and then lysed in ten volumes of NP40 buffer as previously described (Lin et al., 2014a (link)). The primary antibodies and concentrations used for Western blotting were following: KEAP1 (Santa Cruz, sc-365626, 1:1000 dilution), G6PD (1:1,000, Cell Signaling Technology, no. 12263 S), vinculin (1:5,000, Sigma, no. V4505), Myc tag (1:2,000, Cell Signaling Technology, no. 2276 S), SLC7A11 (1:2,000, Cell Signaling Technology, no. 12691 S), GLUT1 (1:1,000, Thermo Fisher, no. PA5-16793), GLUT3 (1:1,000, Santa Cruz, no. sc-74497), NRF2 (1:1,000, Cell Signaling Technology, no. 12721 S).
Koppula P., Olszewski K., Zhang Y., Kondiparthi L., Liu X., Lei G., Das M., Fang B., Poyurovsky M.V, & Gan B. (2021). KEAP1 deficiency drives glucose dependency and sensitizes lung cancer cells and tumors to GLUT inhibition. iScience, 24(6), 102649.
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9

Lentiviral Gene Manipulation in Cells

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Cells were infected with shRNAs for vector control (shCtrl, Open Biosystems), Glut3 (Santa Cruz Biotechnology), β3 and PAK4 (Open Biosystems) or YAP/TAZ (provided by Dr. K-L Guan) using a lentiviral system. pLENTIβ3 was obtained by subcloning the human β3 cDNA of pENTRβ3 vector in the pLENTI expression vector. pRETROYAP was kindly provided by Dr. K-L Guan. Gene silencing or overexpressing was confirmed by either immunoblot analysis or qPCR analysis.
Cosset É., Ilmjärv S., Dutoit V., Elliott K., von Schalscha T., Camargo M.F., Reiss A., Moroishi T., Seguin L., Gomez G., Moo J.S., Preynat-Seauve O., Krause K.H., Chneiweiss H., Sarkaria J.N., Guan K.L., Dietrich P.Y., Weis S.M., Mischel P.S, & Cheresh D.A. (2017). Glut3 addiction is a druggable vulnerability for a molecularly defined subpopulation of glioblastoma. Cancer cell, 32(6), 856-868.e5.
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10

Quantification of Glucose Transporters in Murine Brain

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Mice were decapitated, after 6h of fasting, and their brains were quickly removed and submerged in ice-cold artificial cerebrospinal fluid. Subsequently, about 350 mg of brain tissue was homogenized in Buffer A (Hepes pH 7.9 10 mM, KCl 10 mM, EDTA 0.1 mM, EGTA 0.1 mM, DTT 1mM, PMSF 0.5 mM). Nuclear and cytoplasmic extracts were prepared as previously described [52] (link). For western blot analysis, cytoplasmic proteins (50 μg) were resolved on 10% SDS-PAGE, transferred to nitrocellulose membrane (0.2 μm Bio-rad cat.1620112). Membranes were first immersed for 2 h in blocking solution (5% non-fat dry milk), then probed with antibodies raised against GLUT-1 (Novus Bio, Centennials, CO) and GLUT-3 (Santa Cruz Biotechnology, Santa Cruz, CA). After overnight incubation at 4°C, goatanti rabbit IgG horseradish (Dako) and anti-mouse IgG horseradish (Dako) conjugates were added for 1 h at room temperature, and immune complexes were visualized by enhanced chemiluminescence (ECL, Amersham). Western blots were normalized to beta-actin (GLUT-1) or tubulin (GLUT-3), as loading control proteins, and densitometric analysis was performed using ImageJ software (National Institutes of Health, Bethesda, Maryland, USA).
Nesci V., Russo E., Arcidiacono B., Citraro R., Tallarico M., Constanti A., Brunetti A., De Sarro G, & Leo A. (2020). Metabolic Alterations Predispose to Seizure Development in High-Fat Diet-Treated Mice: the Role of Metformin. Molecular neurobiology, 57(11).
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