Ab652

Manufactured by Abcam

Sourced in United Kingdom, United States

Ab652 is a laboratory equipment product manufactured by Abcam. It is designed for use in scientific research applications. The core function of Ab652 is to provide a tool for researchers to conduct their experiments.

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GLUT1 (ab652) (3 citations)

39 protocols using ab652

Insulin Signaling Pathway Regulation

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3-isobutyl-1-methylxanthine (IBMX, I5879), dexamethasone (D8893), human insulin solution (I9278), antibody to hypoxia-inducible factor (HIF-1α, H6536) and sodium sulfite (Na₂SO₃, S0505) were obtained from Sigma-Aldrich. Antibodies against glucose transporter 1 (Glut1; ab652), glucose transporter 4 (Glut4; ab654), Akt (ab8805), S6K (ab9366), phosphorylated Akt Thr308 (pAkt, ab38449), phosphorylated pyruvate dehydrogenase E1 Ser293 (pPDHE1, ab92696), and β-actin (ab6276) were obtained from Abcam (Cambridge, UK). Antibodies to pyruvate dehydrogenase E1 (PDHE1, sc-65242), insulin receptor β (IRβ, sc-711), insulin receptor substrate-1 (IRS-1, sc-7200), GSK-3β (sc-7291), SREBP1 (sc-13551) and phosphorylated GSK3β (pGSK3β; sc-11757-R) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Antibody to phosphorylated AktSer473 was purchased from Calbiochem (Gibbstown, NJ). Antibodies to AMPK (#2603), phosphorylated AMPKα (Thr172, pAMPK, #2535), and phosphorylated acetyl CoA carboxylase (pACC, #3661) were purchased from Cell Signaling (Beverly, MA). Antibody to phosphorylated IRS-1 Ser-307 (28863) was purchased from Upstate Biotechnology (Canastota, NY). PPARγ (MAB3872) antibody was from EMD Millipore (Billerica, MA, USA).

Lu H., Gao Z., Zhao Z., Weng J, & Ye J. (2015). Transient hypoxia reprograms differentiating adipocytes for enhanced insulin sensitivity and triglyceride accumulation. International journal of obesity (2005), 40(1), 121-128.

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Quantification of Glucose Transporters and Glycolytic Enzymes

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Cells were lysed in radio immunoprecipitation assay buffer (KeyGen, Nanjing, China) containing protease inhibitor PMSF (KeyGen, Nanjing, China) and analyzed for total protein concentration. Western blotting was obtained utilizing 20–40 μg of lysate protein according to the standard protocol. The following antibodies were used in this study: GLUT1 (Abcam, ab652, 1:1,000 dilution), GLUT2 (Abcam, ab54460, 1:1,000 dilution), GLUT3 (Abcam, ab41525, 1:1,000 dilution), GLUT4 (Abcam, ab654, 1:1,000 dilution), GLUT5 (Abcam, ab36057, 1:1,000 dilution), HK2 (Cell Signaling Technology, 2867, 1:1,000 dilution), ALDOC (Abcam, ab87122, 1:1,000 dilution), ENO1 (Proteintech, 11204-1-AP, 1:1,000 dilution), PKM (Abcam, ab38237, 1:1,000 dilution), LDH (Cell Signaling Technology, 3582, 1:1,000 dilution), ACTIN (Abcam, ab15265, 1:1,000 dilution), and E2F1 (Santa Cruz Biotechnology, sc-251, 1:1,000 dilution).

Luo J., Wang H., Wang L., Wang G., Yao Y., Xie K., Li X., Xu L., Shen Y, & Ren B. (2021). lncRNA GAS6-AS1 inhibits progression and glucose metabolism reprogramming in LUAD via repressing E2F1-mediated transcription of GLUT1. Molecular Therapy. Nucleic Acids, 25, 11-24.

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Western Blot Analysis of Signaling Proteins

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Cells were washed in phosphate-buffered saline (PBS) and lysed using radioimmunoprecipitation assay buffer (Invitrogen, Carlsbad, CA) supplemented with a protease inhibitor cocktail (Roche, Pleasanton, CA, USA). The protein concentration was evaluated using a bicinchoninic acid protein assay kit (Bioswamp, PAB180007, Wuhan, China). Equivalent amounts of proteins (30 μg) from each sample were subjected to sodium dodecyl-polyacrylamide electrophoresis, transferred to a polyvinylidene fluoride membrane, blocked in 5% fat-free milk for 2 hours at room temperature, and incubated with the following primary antibodies: PTEN (ab32199, 1:10,000, abcam), PI3K (ab191606, 1:10,000, abcam), p-PI3K (ab182651, 1:10,000, abcam), mTOR (ab32028, 1:10,000, abcam), p-mTOR (ab109268, 1:10,000, abcam), GLUT1 (ab652, 1:10,000, abcam), LDHB (ab85319, 1:10,000, abcam), HK2 (ab209847, 1:10,000, abcam), PKM2 (ab150377, 1:10,000, abcam), or GAPDH (PAB36264, 1:10,000, Bioswamp). Then, the membranes were washed with Tris-buffered saline and incubated with goat anti-rabbit IgG secondary antibody (SAB43711, 1:10,000, Bioswamp) for 2 h at room temperature. An enhanced chemiluminescence kit (Pierce) was used to detect specific bands and autoradiograms were quantified by densitometry (Quantity One software, Bio-Rad, Hercules, CA, USA) using GAPDH as a control. For each group, the quantification was triplicated.

Tian X., Liu M., Huang X., Zhu Q., Liu W., Chen W., Zou Y., Cai Y., Huang S., Chen A., Zhan T., Huang M., Chen X., Han Z, & Tan J. (2020). Noscapine Induces Apoptosis in Human Colon Cancer Cells by Regulating Mitochondrial Damage and Warburg Effect via PTEN/PI3K/mTOR Signaling Pathway. OncoTargets and therapy, 13, 5419-5428.

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Protein extraction and western blot

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To extract total protein, sodium dodecyl sulfate (SDS) lysis buffer (1% SDS, 60 mM Tris-HCl) with protease inhibitor (Roche, Basel, Switzerland) and phosphatase inhibitor (GenDEPOT, Katy, TX, USA) was used. WB was performed as previously reported [28 (link)]. The primary antibodies used were as follows: hexokinase 2 (HK2, ab209847, EPR20839, 1:500), glucose transporter 1(GLUT1, ab652, 1:2000), L-type amino acid transporter 1 (LAT1, ab208776, EPR17573, 1:1000), vascular endothelial growth factor B (VEGFB, ab110649, EPR4555, 1:1000), VEGFC (ab135506, 1:2000), CD133 (ab19898, 1:1000) and Snail/Slug (ab180714, 1:1000) from Abcam (Cambridge, UK), signal transducer and activator of transcription 3 (STAT3, #9139, 1:1000), pSTAT3 (#9145, D3A7, 1:2000) from Cell Signaling Technology (Danvers, MA, USA), and β-actin-HRP (sc-47778, C4, 1:5000) from Santa Cruz Biotechnology (Dalla, TX, USA). For the digital visualization of the chemiluminescent WB, the ChemiDoc XRS (Bio-Rad, Hercules, CA, USA) was used. These experiments were replicated at least three times with similar results. ImageJ (National Institutes of Health, Bethesda, MD, USA) and BioRad Image Lab 6 (Bio-Rad) were used for band quantification.

Park Y., Han Y., Kim D., Cho S., Kim W., Hwang H., Lee H.W., Han D.H., Kim K.S., Yun M, & Lee M. (2022). Impact of Exogenous Treatment with Histidine on Hepatocellular Carcinoma Cells. Cancers, 14(5), 1205.

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Western Blot Analysis of Stem Cell Markers

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Cell samples were lysed in RIPA buffer on ice for 20 min. The lysed cells were collected and centrifuged at 14,000 x g to remove cell debris. Protein concentrations were determined with the BCA Protein Assay Kit (P0009, Beyotime). Each sample containing 30 μg of total protein was separated by SDS-PAGE in a 10% gel and transferred onto PVDF membranes (EMD Millipore Corporation, US). After blockage with 5% nonfat dry milk in Tris-buffered saline with 1‰ Tween (TBST), the membranes were incubated overnight at 4 °C with primary antibodies against brachyury (ab209665, Abcam), GLUT-1 (ab652, Abcam), SHH (sc-365,112, Santa Cruz), CD24 (ab64064, Abcam), KRT-8 (sc-8020, Santa Cruz), HIF-1α (NB100–105, Novus), NOTCH1 (4380, CST), JAGGED1 (ab109536, Abcam), HES1 (11,988, CST), and β-actin (CW0096S, CW Biotech). After three washes with TBST, the membrane was incubated with horseradish peroxidase–conjugated secondary antibodies (Jackson). The protein expression level was determined by densitometric analysis and was normalized to the level of β-actin.

Liu Z., Zheng Z., Qi J., Wang J., Zhou Q., Hu F., Liang J., Li C., Zhang W, & Zhang X. (2018). CD24 identifies nucleus pulposus progenitors/notochordal cells for disc regeneration. Journal of Biological Engineering, 12, 35.

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Western Blotting Protein Detection

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Immunoblotting was performed as described previously^{6 (link)}. Primary antibodies were followed by mouse- or rabbit-conjugated horseradish peroxidase (HRP). HRP-conjugated antibodies (anti-mouse or anti-rabbit IgG HRP conjugate, Promega) were detected by enhanced chemiluminescence detection (Thermofisher). This included the following antibodies: Akt (9272, Cell Signaling), Cpt1a (15184-1-AP, Proteintech group), Hif-1α (10006421, Cayman Chemical Company), phosphor-Akt (4060, Cell Signaling), PIK3cγ (ab140310, abcam), phosphor-mTOR (5536, Cell Signaling), PTEN (9552, Cell Signaling), phosphor-p70 S6K (9204, Cell Signaling). Alternatively, primary antibodies were followed by fluorescently labeled anti-mouse or rabbit antibodies (LiCor) and imaged using the Odyssey infrared imaging system (LiCor). This included the following antibodies: Glut1 (ab652, Abcam), hexokinase 2 (2867, Cell Signaling), hexokinase 1 (ab104835, Abcam), cytochrome C (556432, BD Biosciences), β-actin (A5441, Sigma) and phosphor-S6 (4858, Cell Signaling). All primary antibodies were used at a 1:1,000 dilution, with secondary HRP antibodies used at 1:8000. Secondary LiCor antibodies were used at 1:10000 (anti-mouse) or 1:8000 (anti-rabbit).

Gerriets V.A., Kishton R.J., Johnson M.O., Cohen S., Siska P.J., Nichols A.G., Warmoes M.O., de Cubas A.A., MacIver N.J., Locasale J.W., Turka L.A., Wells A.D, & Rathmell J.C. (2016). Foxp3 and Toll-like receptor signaling balance Treg cell anabolic metabolism for suppression. Nature immunology, 17(12), 1459-1466.

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Quantifying Protein Expression Levels

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The GLUT1, FASN, and matrix metalloproteinase-2 (MMP2) expression levels were measured using Western blot analysis. Samples were lysed with a lysis buffer containing a protease inhibitor cocktail (G2006, Servicebio), and the protein concentrations were determined using a protein assay kit (G2006, Servicebio). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed with 20 μg of protein in each sample using 4% to 15% Mini Protean TGX precast gels (Servicebio). The following primary antibodies were used: anti-GLUT1 (AB652, 1:1000, Abcam), anti-FASN (AB22759, 1:1000, Abcam), and anti-MMP2 (GB11130, 1:1000, Servicebio). The bands were detected using a Western blotting detection system (Epson, Japan) and band intensity was calculated via densitometry using AlphaEaseFC (Alpha Innotech, USA).

Zhao J., Zhang Z., Nie D., Ma H., Yuan G., Su S., Liu S., Liu S, & Tang G. (2019). PET Imaging of Hepatocellular Carcinomas: 18F-Fluoropropionic Acid as a Complementary Radiotracer for 18F-Fluorodeoxyglucose. Molecular Imaging, 18, 1536012118821032.

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Protein Expression Analysis by Western Blot

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Tissue and cellular proteins were extracted using RIPA total protein lysate (Aspen, China) following the manufacturer’s instructions. Proteins from each sample were separated by electrophoresis on 8–15% SDS-PAGE gels and transferred onto PVDF membranes (Aspen). After incubation with WB-specific blocking solution (5% skimmed milk powder (AS1033, Aspen, China) diluted in TBST), the PVDF membranes were separately incubated with anti-GLUT1 (1:1000, ab652, Abcam), anti-GLUT4 (1:2000, ab654, Abcam), anti-(extracellular) GLUT3 (1:1000, AGT-023, Alomone), anti-Bax (1:800, sc-7480, Santa Cruz), anti-Bcl-2 (1:800, sc-7382, Santa Cruz), anti-caspase-3 (cleaved) (1:100, AB3623, Millipore), anti-poly (ADP-ribose) polymerase (PARP) (cleaved) (1:1000, 5625, Cell Signaling Technology), and anti-β-actin (1:10,000, Aspen) antibodies at 4 °C overnight. After the blots were washed, they were incubated with HRP-conjugated secondary antibody for 1.5 h and detected by a chemiluminescent imaging system (Tanon, China).

Zhu J., Zhang Z., Jia J., Wang L., Yang Q., Wang Y, & Chen C. (2019). Sevoflurane Induces Learning and Memory Impairment in Young Mice Through a Reduction in Neuronal Glucose Transporter 3. Cellular and Molecular Neurobiology, 40(6), 879-895.

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Immunohistochemical Analysis of Eyelid Tissues

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Human and mouse eyelid sections were fixed with cold methanol for 15 minutes at −20°C. Following three phosphate-buffered saline (PBS) rinses for 5 minutes each, samples were incubated with 2% bovine serum albumin (BSA, Sigma-Aldrich Corp., St. Louis, MO) in PBS for 60 minutes. The slides were then placed overnight at 4°C in a moist chamber with antibodies specific for pimo (1 : 50; Hypoxyprobe Inc.), Glucose transporter-1 (Glut-1, 1:500, ab652, Abcam, Cambridge, MA), human CD31 (1:500, ab24590, Abcam), Notch1 (1: 10, bTAN20, Developmental Studies Hybridoma Bank, University of Iowa), or cytokeratin 14 (CK14, 1:500, ab181595, Abcam). After 3 additional PBS rinses, donkey anti-rabbit (1:200, ab150075, Abcam,), donkey anti-mouse (1:200, 2492098, EMD Millipore, Temecula, CA), or goat anti-rat (1: 200, a10522, Thermo Fisher Scientific, Grand Island, NY) secondary antibodies were applied the sections for 1 hour at room temperature. The primary antibodies were replaced with mouse or rabbit IgG as negative controls.

Liu Y., Chen D., Chen X., Kam W.R., Hatton M.P, & Sullivan D.A. (2018). Hypoxia: A breath of fresh air for the meibomian gland. The ocular surface, 17(2), 310-317.

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Protein Extraction and Western Blotting

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Cells were washed twice with ice-cold 1 × PBS and lysed using RIPA lysis buffer (Sigma) supplemented with cOmplete, Mini Protease Inhibitor Tablets (1 per 10mL) (Roche) and PhosSTOP phosphatase inhibitor tablets (1 per 10mL). Lysates were sonicated and then centrifuged at 12000 rpm at 4°C for 10 min. Total protein concentration was quantified with pierce BCA protein assay kit (Thermo Scientific™).10 μg of total cell protein were loaded onto 10% SDS-PAGE and then transferred to a microporous polyvinylidene difluoride (PVDF) membrane. Western blotting was performed using Polyclonal anti-GLUT1 antibody (1:2000) (ab652, Abcam), anti-β-actin antibody (Sigma) and goat anti-rabbit or goat anti-mouse IgG-peroxidase conjugate as the secondary antibody. The detection of the proteins was made using chemiluminescence substrate.

Liu R., Fu Z., Zhao M., Gao X., Li H., Mi Q., Liu P., Yang J., Yao Z, & Gao Q. (2017). GLUT1-mediated selective tumor targeting with fluorine containing platinum(II) glycoconjugates. Oncotarget, 8(24), 39476-39496.

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