hglut2 antibody  (R&D Systems)

Journal: Journal of Biological Chemistry

Article Title: Mutations in SLC2A2 Gene Reveal hGLUT2 Function in Pancreatic β Cell Development

doi: 10.1074/jbc.m113.469189

Figure Lengend Snippet: FIGURE 2. Expression and transport function of two SNP variants of hGLUT2. A, expression of hGLUT2 wild type (WT) and two SNP variants (P68L and T110I) in mhAT3F cells. Cells transfected with a pCMV-hGLUT2-HA-IRES-hrGFP construct are identified by the expression of the GFP. Plasma membrane location of hGLUT2 (red in the left panels and white in the right panels) is revealed with an antibody to an extracellular epitope of hGLUT2 in nonpermeabilized cells. Nuclei are stained with DAPI (blue). Scale bar corresponds to 25 m. B, Western blot analysis of membrane fractions from mhAT3F cells transfected or not transfected (NT) with different hGLUT2 constructs. hGLUT2 expression is revealed with an antibody to HA epitope. E-cadherin is used as a loading control of membrane fractions. C, membrane expression of WT, P68L, and T110I hGLUT2 in Xenopus oocytes injected with the corresponding cRNAs. D, dose-response curves of 2-DOG uptake by Xenopus oocytes injected with WT, P68L, or T110I hGLUT2 cRNA. Curves are fitted up using Michaelis-Menten nonlinear regression.

Article Snippet: Surface labeling of Xenopus oocytes was performed as described (29) using hGLUT2 antibody (MAB 1414; R&D Systems).

Techniques: Expressing, Transfection, Construct, Clinical Proteomics, Membrane, Staining, Western Blot, Control, Injection

Journal: Journal of Biological Chemistry

Article Title: Mutations in SLC2A2 Gene Reveal hGLUT2 Function in Pancreatic β Cell Development

doi: 10.1074/jbc.m113.469189

Figure Lengend Snippet: FIGURE 3. Expression and transport function of four FBS-associated mutants of hGLUT2. A, expression of hGLUT2 wild type (WT) and four FBS-associated mutants (G20D, S242R, P417L, and W444R) in mhAT3F cells. Cells transfected with a pCMV-hGLUT2-HA-IRES-hrGFP construct are identified by the expression of the GFP. Subcellular location of hGLUT2 (red in the left panels and white in the right panels) is revealed with an antibody to HA epitope in permeabilized cells. Nuclei are stained with DAPI (blue). Note that G20D hGLUT2 cannot be detected, and S242R hGLUT2 is not targeted at the plasma membrane. Scale bar corresponds to 25 m. B, Western blot analysis of membrane fractions from mhAT3F cell transfected or not transfected (NT) with different hGLUT2 constructs. hGLUT2expressionisrevealedwithanantibodytoHAepitope.E-cadherinisusedasaloadingcontrolofmembranefractions.NotethatG20DorS242RhGLUT2 cannot be detected in membrane fractions. C, membrane expression of WT, S242R, P417L, and W444R hGLUT2 in Xenopus oocytes injected with the corre- sponding cRNAs. D, noncorrected uptake of 2-DOG by Xenopus oocytes injected with water or injected with WT, S242R, P417L, or W444R hGLUT2 cRNA.

Article Snippet: Surface labeling of Xenopus oocytes was performed as described (29) using hGLUT2 antibody (MAB 1414; R&D Systems).

Techniques: Expressing, Transfection, Construct, Staining, Clinical Proteomics, Membrane, Western Blot, Injection

Journal: Journal of Biological Chemistry

Article Title: Mutations in SLC2A2 Gene Reveal hGLUT2 Function in Pancreatic β Cell Development

doi: 10.1074/jbc.m113.469189

Figure Lengend Snippet: FIGURE 4. Expression and transport functions of three engineered mutants of hGLUT2. A, membrane co-localization of wild type (WT), G20S, F295Y, and L368P hGLUT2 (red) and membrane marker E-cadherin (green) in infected mhAT3F cells analyzed by confocal microscopy. Nuclei are stained with DAPI (blue). Scale bar corresponds to 10 m. B, similar migrating profiles of hGLUT2 WT and mutants (G20S, F295Y, and L368P) in membrane fractions of mhAT3F cells analyzed by Western blot. C, comparable membrane expression of WT, G20S, F295Y, and L368P hGLUT2 in Xenopus oocytes injected with the corresponding cRNAs. D, dose-response curves of 2-DOG uptake by Xenopus oocytes injected with WT, G20S, F295Y, and L368P hGLUT2. Curves are fitted up using Michaelis- Menten nonlinear regression.

Article Snippet: Surface labeling of Xenopus oocytes was performed as described (29) using hGLUT2 antibody (MAB 1414; R&D Systems).

Techniques: Expressing, Membrane, Marker, Infection, Confocal Microscopy, Staining, Western Blot, Injection

Journal: Journal of Biological Chemistry

Article Title: Mutations in SLC2A2 Gene Reveal hGLUT2 Function in Pancreatic β Cell Development

doi: 10.1074/jbc.m113.469189

Figure Lengend Snippet: FIGURE5.ImpactofG20S,F295YandL368PhGLUT2mutantsoninsulinsecretion.A,quantificationbyflowcytometryofplasmamembraneexpressionfor wild type (WT), G20S, F295Y, and L368P hGLUT2 in infected MIN6 cells that were cultured in 0 mM (light gray), 5 mM (dark gray), or 25 mM (black) glucose. Nonpermeabilized cells are labeled with an antibody to an extracellular epitope of hGLUT2. Noninfected cells (NI) are used as negative control. B and C, insulin mRNA levels (B) and protein contents (C) in MIN6 cells expressing WT, G20S, F295Y, or L368P hGLUT2 cultured in 25 mM glucose. D, glucose-induced insulin secretion by MIN6 cells expressing WT, G20S, F295Y, or L368P hGLUT2 in response to 1 mM (light gray), 5 mM (dark gray), or 25 mM (black) glucose. E, basal insulin secretion by MIN6 cells expressing WT, G20S, F295Y, or L368P hGLUT2 in the absence of glucose. ns, no significant differences between WT and mutants. *, p 0.05; **, p 0.01 comparison between WT and mutant hGLUT2 at the same glucose concentration.

Article Snippet: Surface labeling of Xenopus oocytes was performed as described (29) using hGLUT2 antibody (MAB 1414; R&D Systems).

Techniques: Infection, Cell Culture, Labeling, Negative Control, Expressing, Comparison, Mutagenesis, Concentration Assay

Journal: Journal of Biological Chemistry

Article Title: Mutations in SLC2A2 Gene Reveal hGLUT2 Function in Pancreatic β Cell Development

doi: 10.1074/jbc.m113.469189

Figure Lengend Snippet: FIGURE 6. G20S, F295Y, and L368P hGLUT2 mutants increase pancreatic cell differentiation. A, immunohistology analysis of rat pancreases cultured for 7 days in the absence (left panels) or presence (middle and right panels) of 10 mM glucose. Staining of insulin (red), amylase (green, left and middle panels), or glucagon (green, right panels) and nuclei (blue) in noninfected pancreas (NI), pancreas infected with an adenoviral vector encoding hGLUT2 wild type (WT), G20S, F295Y, or L368P. Scale bar corresponds to 100 m. B–D, quantification of the surfaces occupied by insulin-positive (B), glucagon-positive (C), or amylase-positive (D) cells expressed as a percentage of total cell surface after 7 days of culture in 0 mM (white bars) or 10 mM glucose (gray bars) glucose. E, proliferative index of insulin-positive cells after 7 days of culture in 0 mM (white bars) or 10 mM (gray bars) glucose, as assayed by the frequency of BrdU-positive nuclei among insulin-positive cells. ##, p 0.01. ns, nonsignificant, comparison between 10 and 0 mM glucose concentrations. **, p 0.01 comparison between WT and mutant hGLUT2 at the same glucose concentration.

Article Snippet: Surface labeling of Xenopus oocytes was performed as described (29) using hGLUT2 antibody (MAB 1414; R&D Systems).

Techniques: Cell Differentiation, Cell Culture, Staining, Infection, Plasmid Preparation, Comparison, Mutagenesis, Concentration Assay

Journal: Journal of Biological Chemistry

Article Title: Mutations in SLC2A2 Gene Reveal hGLUT2 Function in Pancreatic β Cell Development

doi: 10.1074/jbc.m113.469189

Figure Lengend Snippet: FIGURE 8. F295Y hGLUT2 mutant increases pancreatic cell differentia- tion through ChREBP activation. A, immunohistology analysis of rat pan- creases cultured for 7 days in the absence of glucose. Staining of insulin (red), amylase (green), and nuclei (blue) in pancreas infected with an adenoviral vector coding either a dominant-negative form of ChREBP (DN ChREBP), the F295Y hGLUT2 mutant, or both. Scale bar corresponds to 100 m. B, quanti- fication of the surfaces occupied by insulin-positive cells expressed as a per- centageoftotalcellsurfaceafter7daysofculturein0mMglucose.**,p0.01 comparison with NI.

Article Snippet: Surface labeling of Xenopus oocytes was performed as described (29) using hGLUT2 antibody (MAB 1414; R&D Systems).

Techniques: Mutagenesis, Activation Assay, Cell Culture, Staining, Infection, Plasmid Preparation, Dominant Negative Mutation, Comparison

Journal: Journal of Biological Chemistry

Article Title: Mutations in SLC2A2 Gene Reveal hGLUT2 Function in Pancreatic β Cell Development

doi: 10.1074/jbc.m113.469189

Figure Lengend Snippet: FIGURE 7. Pancreatic cells induced by G20S, F295Y, and L368P hGLUT2 mutants secrete insulin in response to glucose. A, glucose-induced insulin secretion by rat pancreases noninfected (NI) or infected with an adenoviral vector encoding hGLUT2 G20S, F295Y, or L368P. **, p 0.01 comparison between NI and mutant hGLUT2 at the same glucose concentration. B and C, morphological studies by electron microscopy of rat pancreases noninfected (NI) or infected with an adenoviral vector coding G20S hGLUT2 mutant after 7 days in culture. Type A cells (very dense core granules) and type B cells (dense core granules with halo) are both visible (3,400). Scale bar corresponds to 1 m. D, immunogold staining of insulin producing cells in a rat pancreas infected with an adenoviral vector coding G20S hGLUT2 mutant. Most of the secretory granules show labeling on the amorphous dense cores (16,000). Scale bar corresponds to 1 m. E, measurement of the diameter of 600 gran- ules of insulin producing cells, using images at a magnification of 12,500. ***, p 0.001 comparison with NI.

Article Snippet: Surface labeling of Xenopus oocytes was performed as described (29) using hGLUT2 antibody (MAB 1414; R&D Systems).

Techniques: Infection, Plasmid Preparation, Comparison, Mutagenesis, Concentration Assay, Electron Microscopy, Staining, Labeling

Journal: Journal of Biological Chemistry

Article Title: Mutations in SLC2A2 Gene Reveal hGLUT2 Function in Pancreatic β Cell Development

doi: 10.1074/jbc.m113.469189

Figure Lengend Snippet: FIGURE 2. Expression and transport function of two SNP variants of hGLUT2. A, expression of hGLUT2 wild type (WT) and two SNP variants (P68L and T110I) in mhAT3F cells. Cells transfected with a pCMV-hGLUT2-HA-IRES-hrGFP construct are identified by the expression of the GFP. Plasma membrane location of hGLUT2 (red in the left panels and white in the right panels) is revealed with an antibody to an extracellular epitope of hGLUT2 in nonpermeabilized cells. Nuclei are stained with DAPI (blue). Scale bar corresponds to 25 m. B, Western blot analysis of membrane fractions from mhAT3F cells transfected or not transfected (NT) with different hGLUT2 constructs. hGLUT2 expression is revealed with an antibody to HA epitope. E-cadherin is used as a loading control of membrane fractions. C, membrane expression of WT, P68L, and T110I hGLUT2 in Xenopus oocytes injected with the corresponding cRNAs. D, dose-response curves of 2-DOG uptake by Xenopus oocytes injected with WT, P68L, or T110I hGLUT2 cRNA. Curves are fitted up using Michaelis-Menten nonlinear regression.

Article Snippet: Cell suspensions were incubated for 1 h with anti-hGLUT2 conjugated to phycoerythrin antibody (directed against extracellular epitope of hGLUT2, FAB1414P; R&D Systems), diluted in binding buffer.

Techniques: Expressing, Transfection, Construct, Clinical Proteomics, Membrane, Staining, Western Blot, Control, Injection

Journal: Journal of Biological Chemistry

Article Title: Mutations in SLC2A2 Gene Reveal hGLUT2 Function in Pancreatic β Cell Development

doi: 10.1074/jbc.m113.469189

Figure Lengend Snippet: FIGURE 3. Expression and transport function of four FBS-associated mutants of hGLUT2. A, expression of hGLUT2 wild type (WT) and four FBS-associated mutants (G20D, S242R, P417L, and W444R) in mhAT3F cells. Cells transfected with a pCMV-hGLUT2-HA-IRES-hrGFP construct are identified by the expression of the GFP. Subcellular location of hGLUT2 (red in the left panels and white in the right panels) is revealed with an antibody to HA epitope in permeabilized cells. Nuclei are stained with DAPI (blue). Note that G20D hGLUT2 cannot be detected, and S242R hGLUT2 is not targeted at the plasma membrane. Scale bar corresponds to 25 m. B, Western blot analysis of membrane fractions from mhAT3F cell transfected or not transfected (NT) with different hGLUT2 constructs. hGLUT2expressionisrevealedwithanantibodytoHAepitope.E-cadherinisusedasaloadingcontrolofmembranefractions.NotethatG20DorS242RhGLUT2 cannot be detected in membrane fractions. C, membrane expression of WT, S242R, P417L, and W444R hGLUT2 in Xenopus oocytes injected with the corre- sponding cRNAs. D, noncorrected uptake of 2-DOG by Xenopus oocytes injected with water or injected with WT, S242R, P417L, or W444R hGLUT2 cRNA.

Article Snippet: Cell suspensions were incubated for 1 h with anti-hGLUT2 conjugated to phycoerythrin antibody (directed against extracellular epitope of hGLUT2, FAB1414P; R&D Systems), diluted in binding buffer.

Techniques: Expressing, Transfection, Construct, Staining, Clinical Proteomics, Membrane, Western Blot, Injection

Journal: Journal of Biological Chemistry

Article Title: Mutations in SLC2A2 Gene Reveal hGLUT2 Function in Pancreatic β Cell Development

doi: 10.1074/jbc.m113.469189

Figure Lengend Snippet: FIGURE 4. Expression and transport functions of three engineered mutants of hGLUT2. A, membrane co-localization of wild type (WT), G20S, F295Y, and L368P hGLUT2 (red) and membrane marker E-cadherin (green) in infected mhAT3F cells analyzed by confocal microscopy. Nuclei are stained with DAPI (blue). Scale bar corresponds to 10 m. B, similar migrating profiles of hGLUT2 WT and mutants (G20S, F295Y, and L368P) in membrane fractions of mhAT3F cells analyzed by Western blot. C, comparable membrane expression of WT, G20S, F295Y, and L368P hGLUT2 in Xenopus oocytes injected with the corresponding cRNAs. D, dose-response curves of 2-DOG uptake by Xenopus oocytes injected with WT, G20S, F295Y, and L368P hGLUT2. Curves are fitted up using Michaelis- Menten nonlinear regression.

Article Snippet: Cell suspensions were incubated for 1 h with anti-hGLUT2 conjugated to phycoerythrin antibody (directed against extracellular epitope of hGLUT2, FAB1414P; R&D Systems), diluted in binding buffer.

Techniques: Expressing, Membrane, Marker, Infection, Confocal Microscopy, Staining, Western Blot, Injection

Journal: Journal of Biological Chemistry

Article Title: Mutations in SLC2A2 Gene Reveal hGLUT2 Function in Pancreatic β Cell Development

doi: 10.1074/jbc.m113.469189

Figure Lengend Snippet: FIGURE5.ImpactofG20S,F295YandL368PhGLUT2mutantsoninsulinsecretion.A,quantificationbyflowcytometryofplasmamembraneexpressionfor wild type (WT), G20S, F295Y, and L368P hGLUT2 in infected MIN6 cells that were cultured in 0 mM (light gray), 5 mM (dark gray), or 25 mM (black) glucose. Nonpermeabilized cells are labeled with an antibody to an extracellular epitope of hGLUT2. Noninfected cells (NI) are used as negative control. B and C, insulin mRNA levels (B) and protein contents (C) in MIN6 cells expressing WT, G20S, F295Y, or L368P hGLUT2 cultured in 25 mM glucose. D, glucose-induced insulin secretion by MIN6 cells expressing WT, G20S, F295Y, or L368P hGLUT2 in response to 1 mM (light gray), 5 mM (dark gray), or 25 mM (black) glucose. E, basal insulin secretion by MIN6 cells expressing WT, G20S, F295Y, or L368P hGLUT2 in the absence of glucose. ns, no significant differences between WT and mutants. *, p 0.05; **, p 0.01 comparison between WT and mutant hGLUT2 at the same glucose concentration.

Article Snippet: Cell suspensions were incubated for 1 h with anti-hGLUT2 conjugated to phycoerythrin antibody (directed against extracellular epitope of hGLUT2, FAB1414P; R&D Systems), diluted in binding buffer.

Techniques: Infection, Cell Culture, Labeling, Negative Control, Expressing, Comparison, Mutagenesis, Concentration Assay

Journal: Journal of Biological Chemistry

Article Title: Mutations in SLC2A2 Gene Reveal hGLUT2 Function in Pancreatic β Cell Development

doi: 10.1074/jbc.m113.469189

Figure Lengend Snippet: FIGURE 6. G20S, F295Y, and L368P hGLUT2 mutants increase pancreatic cell differentiation. A, immunohistology analysis of rat pancreases cultured for 7 days in the absence (left panels) or presence (middle and right panels) of 10 mM glucose. Staining of insulin (red), amylase (green, left and middle panels), or glucagon (green, right panels) and nuclei (blue) in noninfected pancreas (NI), pancreas infected with an adenoviral vector encoding hGLUT2 wild type (WT), G20S, F295Y, or L368P. Scale bar corresponds to 100 m. B–D, quantification of the surfaces occupied by insulin-positive (B), glucagon-positive (C), or amylase-positive (D) cells expressed as a percentage of total cell surface after 7 days of culture in 0 mM (white bars) or 10 mM glucose (gray bars) glucose. E, proliferative index of insulin-positive cells after 7 days of culture in 0 mM (white bars) or 10 mM (gray bars) glucose, as assayed by the frequency of BrdU-positive nuclei among insulin-positive cells. ##, p 0.01. ns, nonsignificant, comparison between 10 and 0 mM glucose concentrations. **, p 0.01 comparison between WT and mutant hGLUT2 at the same glucose concentration.

Article Snippet: Cell suspensions were incubated for 1 h with anti-hGLUT2 conjugated to phycoerythrin antibody (directed against extracellular epitope of hGLUT2, FAB1414P; R&D Systems), diluted in binding buffer.

Techniques: Cell Differentiation, Cell Culture, Staining, Infection, Plasmid Preparation, Comparison, Mutagenesis, Concentration Assay

Journal: Journal of Biological Chemistry

Article Title: Mutations in SLC2A2 Gene Reveal hGLUT2 Function in Pancreatic β Cell Development

doi: 10.1074/jbc.m113.469189

Figure Lengend Snippet: FIGURE 8. F295Y hGLUT2 mutant increases pancreatic cell differentia- tion through ChREBP activation. A, immunohistology analysis of rat pan- creases cultured for 7 days in the absence of glucose. Staining of insulin (red), amylase (green), and nuclei (blue) in pancreas infected with an adenoviral vector coding either a dominant-negative form of ChREBP (DN ChREBP), the F295Y hGLUT2 mutant, or both. Scale bar corresponds to 100 m. B, quanti- fication of the surfaces occupied by insulin-positive cells expressed as a per- centageoftotalcellsurfaceafter7daysofculturein0mMglucose.**,p0.01 comparison with NI.

Article Snippet: Cell suspensions were incubated for 1 h with anti-hGLUT2 conjugated to phycoerythrin antibody (directed against extracellular epitope of hGLUT2, FAB1414P; R&D Systems), diluted in binding buffer.

Techniques: Mutagenesis, Activation Assay, Cell Culture, Staining, Infection, Plasmid Preparation, Dominant Negative Mutation, Comparison

Journal: Journal of Biological Chemistry

Article Title: Mutations in SLC2A2 Gene Reveal hGLUT2 Function in Pancreatic β Cell Development

doi: 10.1074/jbc.m113.469189

Figure Lengend Snippet: FIGURE 7. Pancreatic cells induced by G20S, F295Y, and L368P hGLUT2 mutants secrete insulin in response to glucose. A, glucose-induced insulin secretion by rat pancreases noninfected (NI) or infected with an adenoviral vector encoding hGLUT2 G20S, F295Y, or L368P. **, p 0.01 comparison between NI and mutant hGLUT2 at the same glucose concentration. B and C, morphological studies by electron microscopy of rat pancreases noninfected (NI) or infected with an adenoviral vector coding G20S hGLUT2 mutant after 7 days in culture. Type A cells (very dense core granules) and type B cells (dense core granules with halo) are both visible (3,400). Scale bar corresponds to 1 m. D, immunogold staining of insulin producing cells in a rat pancreas infected with an adenoviral vector coding G20S hGLUT2 mutant. Most of the secretory granules show labeling on the amorphous dense cores (16,000). Scale bar corresponds to 1 m. E, measurement of the diameter of 600 gran- ules of insulin producing cells, using images at a magnification of 12,500. ***, p 0.001 comparison with NI.

Article Snippet: Cell suspensions were incubated for 1 h with anti-hGLUT2 conjugated to phycoerythrin antibody (directed against extracellular epitope of hGLUT2, FAB1414P; R&D Systems), diluted in binding buffer.

Techniques: Infection, Plasmid Preparation, Comparison, Mutagenesis, Concentration Assay, Electron Microscopy, Staining, Labeling