Human Glut1 Antibody

Detection of Human Glut1 by Flow Cytometry

TCR stimulation results in Glut-1 expression and concomitant glucose uptake in CD4 and CD8 lymphocytes: Induction of H1RBD and H2RBD binding. CD4+ and CD8+ T lymphocytes were isolated by negative selection and stimulated via the TCR using alpha CD3/ alpha CD28 mAbs. (A) Non-activated and TCR-activated T cells were used for binding assays with mAb1418 followed by incubation with a FITC-conjugated alpha mouse IgG. Intracellular Glut-1 levels were monitored in permeabilized cells using the C-term Glut-1 polyclonal antibody followed by incubation with a FITC-conjugated sheep alpha rabbit IgG antibody. Filled histograms depict binding in the presence of the secondary FITC-conjugated antibody alone. Expression of the HTLV-1 Env receptor was detected by a 30 min incubation of the non-activated and TCR-activated cells with rabbit rFc-tagged H1RBD fusion protein at 37°C and binding was revealed by a 20 min incubation at 4°C with a FITC-conjugated sheep alpha rabbit IgG antibody. Binding to the H2RBD domain fused directly to EGFP (H2RBD-EGFP) was detected following a 30 min incubation at 37°C. (B) Glucose uptake was assayed by incubating non-activated and TCR-activated CD4 and CD8 T cells (1 × 106) with 2-deoxy-D [1-3H]glucose (2 μCi) for 45 min at 37°C. Uptake for each cell population is expressed as mean counts per minute (CPM) for triplicate samples, error bars indicate SD. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/17504522), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human Glut1 by Flow Cytometry

Endogenous Glut-1 expression in diverse cell types is not reflected by mAb1418 reactivity but correlates with binding of the HTLV-1 and HTLV-2 Env RBDs. (A) 293T, Jurkat and primary human erythrocytes were stained with mAb1418 and control binding with the secondary FITC-conjugated antibody is shown in all histograms (filled). Intracellular Glut-1 levels in permeabilized 293T and Jurkat cells were monitored with mAb1418 as well as the C-term polyclonal Glut-1 antibody. Expression of the HTLV-1 and HTLV-2 receptor was monitored by incubation of cells for 30 min at 37°C with the rFc-tagged H1RBD and H2RBD fusion proteins as well as the H2RBD-EGFP fusion protein. Binding is shown in solid line histograms whereas control immunofluorescence is shown in filled histograms. (B) Total Glut-1 protein levels in cell extracts from 293T, Jurkat and human erythrocytes were monitored by immunoblotting with an anti-C-term Glut-1 antibody. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/17504522), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human Glut1 by Flow Cytometry

Antibody and HRBD binding following transfection of the Glut-1 and Glut-3 glucose transporters. (A) 293T cells were transfected with Glut-1 or Glut-3 expression vectors and assayed for binding to the mAb1418 and C-term anti-Glut-1 polyclonal Ab. The former stainings were performed on whole cells as well as permeabilized cells to determine cell surface and total binding, respectively. All stainings using the anti-Glut-1 pAb were performed on permeabilized cells as the recognized epitope is intracellular. Staining was performed at 4°C. Specific binding and background fluorescence due to the secondary conjugated Ab are indicated in solid line and filled histograms, respectively. (B) Control and transfected 293T cells were incubated with rFc-tagged H1RBD and H2RBD fusion proteins for 30 min at 37°C followed by incubation with a FITC-conjugated alpha rabbit-Fc antibody at 4°C. Direct binding to H2RBD was demonstrated by incubation of cells with an EGFP-tagged envelope (H2RBD-EGFP). Binding is shown in solid line histograms whereas control immunofluorescence is shown in filled histograms. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/17504522), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human Glut1 by Flow Cytometry

Effect of ethanol on CD4+ T cell glucose metabolism. (A) Representative FACS plots of GLUT1 expression in DIFF and DIFF + EtOH CD4+ T cells. (B) Ethanol increased CD4+ T cell GLUT1 expression. (C) Representative FACS histograms of 2-NBDG uptake in DIFF and DIFF + EtOH CD4+ T cells. (D) Ethanol increased glucose uptake within differentiated CD4+ T cells. (E) All experimental groups were analyzed using the Glycolytic Stress test. Ethanol increased glycolysis (F) and glycolytic capacity (G) within differentiated groups. MFI = mean fluorescent intensity, 2-NBDG = 2-Deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose, ECAR = extracellular acidification rate, Naïve = undifferentiated and no ethanol treatment, EtOH = undifferentiated and ethanol-treated, DIFF = differentiated and no ethanol treatment, and DIFF + EtOH = differentiated and ethanol-treated. Data represents average values using CD4+ T cells from 6 donors expressed as mean ± SEM. Significant differences (p<0.05) were determined by repeated measures 2-way ANOVA. *p ≤ 0.05. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/35634279), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human Glut1 by Flow Cytometry

Effect of ethanol on CD4+ T cell glucose metabolism. (A) Representative FACS plots of GLUT1 expression in DIFF and DIFF + EtOH CD4+ T cells. (B) Ethanol increased CD4+ T cell GLUT1 expression. (C) Representative FACS histograms of 2-NBDG uptake in DIFF and DIFF + EtOH CD4+ T cells. (D) Ethanol increased glucose uptake within differentiated CD4+ T cells. (E) All experimental groups were analyzed using the Glycolytic Stress test. Ethanol increased glycolysis (F) and glycolytic capacity (G) within differentiated groups. MFI = mean fluorescent intensity, 2-NBDG = 2-Deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose, ECAR = extracellular acidification rate, Naïve = undifferentiated and no ethanol treatment, EtOH = undifferentiated and ethanol-treated, DIFF = differentiated and no ethanol treatment, and DIFF + EtOH = differentiated and ethanol-treated. Data represents average values using CD4+ T cells from 6 donors expressed as mean ± SEM. Significant differences (p<0.05) were determined by repeated measures 2-way ANOVA. *p ≤ 0.05. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/35634279), licensed under a CC-BY license. Not internally tested by R&D Systems.