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anti glut4  (Alomone Labs)


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    Alomone Labs anti glut4
    a, Schematic for measuring C-peptide and Ins2 mRNA in fed and starved (10–14 h) conditions (left) and C-peptide cleavage during insulin synthesis (right). b, ELISA for C-peptide in blood and protein lysates from retina of fed (ad libitum) and starved mice. Y axis shows percentage of C-peptide found in fed mice. n = 5 mice for fed condition, n = 4 mice for starved. RT–PCR comparing Ins2 expression in RPE from fed or starved mice (right). n = 8 mice per condition. c, RT–PCR measuring Ins2 in RPE from control and Ins2 KO mice. n = 6 mice per genotype. d, Glucose tolerance assay (readout of insulin function) measuring circulating glucose levels over time in WT and Ins2 KO mice starved and then given glucose intraperitoneally (top). n = 4 mice per genotype. ELISA for C-peptide in blood of control and Ins2 KO mice shows no difference between genotypes (bottom). n = 4 mice per genotype. e, Schematic of the IPM (left). IPM was isolated from WT and Ins2 KO mice and the secreted C-peptide in IPM was measured by ELISA (middle). n = 4 mice per genotype. ELISA of C-peptide in lysates of RPE from WT and Ins2 KO mice (right). n = 4 mice WT, n = 3 Ins2 KO. f, Insulin receptor was immunoprecipitated from retina lysates of control and Ins2 KO mice, either fed or starved. InsR tyrosine phosphorylation was assessed using phospho-specific InsR antibodies via immunoblotting and quantified as the ratio of p-InsR to total InsR. Retina lysates were also probed for <t>GLUT4</t> and quantified as the ratio of GLUT4 to actin (normalized to control). n = 4 mice for fed, n = 6 starved conditions. Plots are presented as in Fig. 1. *P ≤ 0.05,**P ≤ 0.01,***P ≤ 0.001, unpaired two-tailed t-test (b) and paired two-tailed t-test (f).
    Anti Glut4, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 92/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Phagocytosis in the retina promotes local insulin production in the eye"

    Article Title: Phagocytosis in the retina promotes local insulin production in the eye

    Journal: Nature metabolism

    doi: 10.1038/s42255-022-00728-0

    a, Schematic for measuring C-peptide and Ins2 mRNA in fed and starved (10–14 h) conditions (left) and C-peptide cleavage during insulin synthesis (right). b, ELISA for C-peptide in blood and protein lysates from retina of fed (ad libitum) and starved mice. Y axis shows percentage of C-peptide found in fed mice. n = 5 mice for fed condition, n = 4 mice for starved. RT–PCR comparing Ins2 expression in RPE from fed or starved mice (right). n = 8 mice per condition. c, RT–PCR measuring Ins2 in RPE from control and Ins2 KO mice. n = 6 mice per genotype. d, Glucose tolerance assay (readout of insulin function) measuring circulating glucose levels over time in WT and Ins2 KO mice starved and then given glucose intraperitoneally (top). n = 4 mice per genotype. ELISA for C-peptide in blood of control and Ins2 KO mice shows no difference between genotypes (bottom). n = 4 mice per genotype. e, Schematic of the IPM (left). IPM was isolated from WT and Ins2 KO mice and the secreted C-peptide in IPM was measured by ELISA (middle). n = 4 mice per genotype. ELISA of C-peptide in lysates of RPE from WT and Ins2 KO mice (right). n = 4 mice WT, n = 3 Ins2 KO. f, Insulin receptor was immunoprecipitated from retina lysates of control and Ins2 KO mice, either fed or starved. InsR tyrosine phosphorylation was assessed using phospho-specific InsR antibodies via immunoblotting and quantified as the ratio of p-InsR to total InsR. Retina lysates were also probed for GLUT4 and quantified as the ratio of GLUT4 to actin (normalized to control). n = 4 mice for fed, n = 6 starved conditions. Plots are presented as in Fig. 1. *P ≤ 0.05,**P ≤ 0.01,***P ≤ 0.001, unpaired two-tailed t-test (b) and paired two-tailed t-test (f).
    Figure Legend Snippet: a, Schematic for measuring C-peptide and Ins2 mRNA in fed and starved (10–14 h) conditions (left) and C-peptide cleavage during insulin synthesis (right). b, ELISA for C-peptide in blood and protein lysates from retina of fed (ad libitum) and starved mice. Y axis shows percentage of C-peptide found in fed mice. n = 5 mice for fed condition, n = 4 mice for starved. RT–PCR comparing Ins2 expression in RPE from fed or starved mice (right). n = 8 mice per condition. c, RT–PCR measuring Ins2 in RPE from control and Ins2 KO mice. n = 6 mice per genotype. d, Glucose tolerance assay (readout of insulin function) measuring circulating glucose levels over time in WT and Ins2 KO mice starved and then given glucose intraperitoneally (top). n = 4 mice per genotype. ELISA for C-peptide in blood of control and Ins2 KO mice shows no difference between genotypes (bottom). n = 4 mice per genotype. e, Schematic of the IPM (left). IPM was isolated from WT and Ins2 KO mice and the secreted C-peptide in IPM was measured by ELISA (middle). n = 4 mice per genotype. ELISA of C-peptide in lysates of RPE from WT and Ins2 KO mice (right). n = 4 mice WT, n = 3 Ins2 KO. f, Insulin receptor was immunoprecipitated from retina lysates of control and Ins2 KO mice, either fed or starved. InsR tyrosine phosphorylation was assessed using phospho-specific InsR antibodies via immunoblotting and quantified as the ratio of p-InsR to total InsR. Retina lysates were also probed for GLUT4 and quantified as the ratio of GLUT4 to actin (normalized to control). n = 4 mice for fed, n = 6 starved conditions. Plots are presented as in Fig. 1. *P ≤ 0.05,**P ≤ 0.01,***P ≤ 0.001, unpaired two-tailed t-test (b) and paired two-tailed t-test (f).

    Techniques Used: Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Control, Isolation, Immunoprecipitation, Western Blot, Two Tailed Test

    a, Representative image of three independent experiments showing immunofluorescence analysis with anti-rhodopsin (magenta) on retina sections 2 h after light onset (peak phagocytosis time) from fed and starved (10–14 h) mice. Sections were counterstained with 4,6-diamidino-2-phenylindole (DAPI) (blue). RPE is outlined in white. b, Flow cytometry-based phagocytosis assay on isolated fixed and permeabilized RPE stained with rhodopsin, from fed or starved mice, obtained 2 h after light onset. Phagocytosis was measured as % of rhodopsin+ RPE within total RPE. n = 3 mice per condition. c, RT–PCR for Ins2 expression in RPE from fed mice at different times of the day. Lights on at 6:00 and lights off at 20:00. n = 8, 7, 7, 7, 3 and 3 mice used for times of 5:00, 6:00, 8:00, 15:00, 20:00 and 1:00, respectively. FC, fold change. d, InsR immunoprecipitated from retina lysates, at different times of day from overnight starved control and Ins2 KO mice, were probed using phospho-specific InsR antibodies. The same retina lysates were probed for GLUT4. Values were normalized to the average of control across all time points. n = 3 mice for each time point. e, Schematic of phagocytic receptors used in POS recognition (left). RT–PCR for Ins2 in RPE isolated from WT and MerTK KO or CD36 KO mice 2 h after light onset (right). n = 10 and 11 mice for WT versus MerTK KO and n = 6 and 5 mice for WT versus CD36 KO. f, Phagocytosis quantification using flow cytometry on isolated RPE from WT and MerTKCR mice (middle). n = 3 mice used per genotype. RT–PCR measuring Ins2 expression in isolated RPE from WT and MerTKCR (right). n = 14 and 10 mice for WT and MerTKCR, respectively. Plots are presented as in Fig. 1. *P ≤ 0.05,**P ≤ 0.01,***P ≤ 0.001, ****P ≤ 0.0001, one-way ANOVA (c), two-way ANOVA with Tukey’s multiple comparisons test (d), unpaired two-tailed t-test (e,f RT–PCR) and paired two-tailed t-test (f phagocytosis quantification).
    Figure Legend Snippet: a, Representative image of three independent experiments showing immunofluorescence analysis with anti-rhodopsin (magenta) on retina sections 2 h after light onset (peak phagocytosis time) from fed and starved (10–14 h) mice. Sections were counterstained with 4,6-diamidino-2-phenylindole (DAPI) (blue). RPE is outlined in white. b, Flow cytometry-based phagocytosis assay on isolated fixed and permeabilized RPE stained with rhodopsin, from fed or starved mice, obtained 2 h after light onset. Phagocytosis was measured as % of rhodopsin+ RPE within total RPE. n = 3 mice per condition. c, RT–PCR for Ins2 expression in RPE from fed mice at different times of the day. Lights on at 6:00 and lights off at 20:00. n = 8, 7, 7, 7, 3 and 3 mice used for times of 5:00, 6:00, 8:00, 15:00, 20:00 and 1:00, respectively. FC, fold change. d, InsR immunoprecipitated from retina lysates, at different times of day from overnight starved control and Ins2 KO mice, were probed using phospho-specific InsR antibodies. The same retina lysates were probed for GLUT4. Values were normalized to the average of control across all time points. n = 3 mice for each time point. e, Schematic of phagocytic receptors used in POS recognition (left). RT–PCR for Ins2 in RPE isolated from WT and MerTK KO or CD36 KO mice 2 h after light onset (right). n = 10 and 11 mice for WT versus MerTK KO and n = 6 and 5 mice for WT versus CD36 KO. f, Phagocytosis quantification using flow cytometry on isolated RPE from WT and MerTKCR mice (middle). n = 3 mice used per genotype. RT–PCR measuring Ins2 expression in isolated RPE from WT and MerTKCR (right). n = 14 and 10 mice for WT and MerTKCR, respectively. Plots are presented as in Fig. 1. *P ≤ 0.05,**P ≤ 0.01,***P ≤ 0.001, ****P ≤ 0.0001, one-way ANOVA (c), two-way ANOVA with Tukey’s multiple comparisons test (d), unpaired two-tailed t-test (e,f RT–PCR) and paired two-tailed t-test (f phagocytosis quantification).

    Techniques Used: Immunofluorescence, Flow Cytometry, Phagocytosis Assay, Isolation, Staining, Reverse Transcription Polymerase Chain Reaction, Expressing, Immunoprecipitation, Control, Two Tailed Test

    a. Quantification of OS phagocytosis by the RPE 2 hours after light onset using immunohistochemistry. Quantification of phagocytosis was measured by the amount of Rhodopsin immunoreactivity in the RPE divided by pixels and presented as phagosomes per area on the y-axis (left). Quantification of the number of phagosomes in RPE was done by counting Rhodopsin puncta in the RPE (right). n = 3 mice used for each condition. *p ≤ .05 paired two-tailed t-test. b. Western blot against Rhodopsin on isolated RPE protein lysates from fed and starved mice at 8 am and 10 am (left). Right panel is quantification of the blot to evaluate POS degradation showing 10 am band intensity as a percent of 8 am (peak phagocytosis) band intensity (right). n = 2 mice used for each time point. c. Insulin receptor was immunoprecipitated from lysates of retina from control and MerTK KO mice that were starved overnight. The lysates were probed for InsR phosphorylation or GLUT4 levels by immunoblotting. C-peptide 2 levels were determined by ELISA. N = 6 mice for each condition. *p < .05 paired two-tailed t-test. d. Schematic of WT and cleavage-resistant ‘gain of function’ MerTKCR mice with altered cleavage sites indicated (left). e. Phagocytosis quantification of ingested photoreceptor outer segments using flow cytometry on isolated RPE stained with antibody against rhodopsin from Control and Ins2 KO mice two hours after light onset. n = 7 mice used for each genotype. Plots are presented as in Fig. 1.
    Figure Legend Snippet: a. Quantification of OS phagocytosis by the RPE 2 hours after light onset using immunohistochemistry. Quantification of phagocytosis was measured by the amount of Rhodopsin immunoreactivity in the RPE divided by pixels and presented as phagosomes per area on the y-axis (left). Quantification of the number of phagosomes in RPE was done by counting Rhodopsin puncta in the RPE (right). n = 3 mice used for each condition. *p ≤ .05 paired two-tailed t-test. b. Western blot against Rhodopsin on isolated RPE protein lysates from fed and starved mice at 8 am and 10 am (left). Right panel is quantification of the blot to evaluate POS degradation showing 10 am band intensity as a percent of 8 am (peak phagocytosis) band intensity (right). n = 2 mice used for each time point. c. Insulin receptor was immunoprecipitated from lysates of retina from control and MerTK KO mice that were starved overnight. The lysates were probed for InsR phosphorylation or GLUT4 levels by immunoblotting. C-peptide 2 levels were determined by ELISA. N = 6 mice for each condition. *p < .05 paired two-tailed t-test. d. Schematic of WT and cleavage-resistant ‘gain of function’ MerTKCR mice with altered cleavage sites indicated (left). e. Phagocytosis quantification of ingested photoreceptor outer segments using flow cytometry on isolated RPE stained with antibody against rhodopsin from Control and Ins2 KO mice two hours after light onset. n = 7 mice used for each genotype. Plots are presented as in Fig. 1.

    Techniques Used: Immunohistochemistry, Two Tailed Test, Western Blot, Isolation, Immunoprecipitation, Control, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Staining



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    Image Search Results


    a, Schematic for measuring C-peptide and Ins2 mRNA in fed and starved (10–14 h) conditions (left) and C-peptide cleavage during insulin synthesis (right). b, ELISA for C-peptide in blood and protein lysates from retina of fed (ad libitum) and starved mice. Y axis shows percentage of C-peptide found in fed mice. n = 5 mice for fed condition, n = 4 mice for starved. RT–PCR comparing Ins2 expression in RPE from fed or starved mice (right). n = 8 mice per condition. c, RT–PCR measuring Ins2 in RPE from control and Ins2 KO mice. n = 6 mice per genotype. d, Glucose tolerance assay (readout of insulin function) measuring circulating glucose levels over time in WT and Ins2 KO mice starved and then given glucose intraperitoneally (top). n = 4 mice per genotype. ELISA for C-peptide in blood of control and Ins2 KO mice shows no difference between genotypes (bottom). n = 4 mice per genotype. e, Schematic of the IPM (left). IPM was isolated from WT and Ins2 KO mice and the secreted C-peptide in IPM was measured by ELISA (middle). n = 4 mice per genotype. ELISA of C-peptide in lysates of RPE from WT and Ins2 KO mice (right). n = 4 mice WT, n = 3 Ins2 KO. f, Insulin receptor was immunoprecipitated from retina lysates of control and Ins2 KO mice, either fed or starved. InsR tyrosine phosphorylation was assessed using phospho-specific InsR antibodies via immunoblotting and quantified as the ratio of p-InsR to total InsR. Retina lysates were also probed for GLUT4 and quantified as the ratio of GLUT4 to actin (normalized to control). n = 4 mice for fed, n = 6 starved conditions. Plots are presented as in Fig. 1. *P ≤ 0.05,**P ≤ 0.01,***P ≤ 0.001, unpaired two-tailed t-test (b) and paired two-tailed t-test (f).

    Journal: Nature metabolism

    Article Title: Phagocytosis in the retina promotes local insulin production in the eye

    doi: 10.1038/s42255-022-00728-0

    Figure Lengend Snippet: a, Schematic for measuring C-peptide and Ins2 mRNA in fed and starved (10–14 h) conditions (left) and C-peptide cleavage during insulin synthesis (right). b, ELISA for C-peptide in blood and protein lysates from retina of fed (ad libitum) and starved mice. Y axis shows percentage of C-peptide found in fed mice. n = 5 mice for fed condition, n = 4 mice for starved. RT–PCR comparing Ins2 expression in RPE from fed or starved mice (right). n = 8 mice per condition. c, RT–PCR measuring Ins2 in RPE from control and Ins2 KO mice. n = 6 mice per genotype. d, Glucose tolerance assay (readout of insulin function) measuring circulating glucose levels over time in WT and Ins2 KO mice starved and then given glucose intraperitoneally (top). n = 4 mice per genotype. ELISA for C-peptide in blood of control and Ins2 KO mice shows no difference between genotypes (bottom). n = 4 mice per genotype. e, Schematic of the IPM (left). IPM was isolated from WT and Ins2 KO mice and the secreted C-peptide in IPM was measured by ELISA (middle). n = 4 mice per genotype. ELISA of C-peptide in lysates of RPE from WT and Ins2 KO mice (right). n = 4 mice WT, n = 3 Ins2 KO. f, Insulin receptor was immunoprecipitated from retina lysates of control and Ins2 KO mice, either fed or starved. InsR tyrosine phosphorylation was assessed using phospho-specific InsR antibodies via immunoblotting and quantified as the ratio of p-InsR to total InsR. Retina lysates were also probed for GLUT4 and quantified as the ratio of GLUT4 to actin (normalized to control). n = 4 mice for fed, n = 6 starved conditions. Plots are presented as in Fig. 1. *P ≤ 0.05,**P ≤ 0.01,***P ≤ 0.001, unpaired two-tailed t-test (b) and paired two-tailed t-test (f).

    Article Snippet: Antibodies used were anti-insulin (Agilent, IR002), anti-insulin (Cell Signaling, 3014), anti-C-peptide (Phoenix Pharmaceuticals, H-035-03), anti-Cre recombinase (Millipore, MAB3120), anti-rhodopsin (Abcam, ab98887), anti-cone arrestin (Millipore, AB15282), anti-phospho insulin receptor-β (Tyr1150/1151) (19H7) (Cell Signaling, 3024), anti-insulin receptor-β (Novus Biologicals, NBP2 12793), anti-Glut4 (Alomone Labs, AGT 024), anti-β actin HRP (Sigma, A3854) and anti-rabbit IgG HRP (GE Healthcare, NA934V).

    Techniques: Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Control, Isolation, Immunoprecipitation, Western Blot, Two Tailed Test

    a, Representative image of three independent experiments showing immunofluorescence analysis with anti-rhodopsin (magenta) on retina sections 2 h after light onset (peak phagocytosis time) from fed and starved (10–14 h) mice. Sections were counterstained with 4,6-diamidino-2-phenylindole (DAPI) (blue). RPE is outlined in white. b, Flow cytometry-based phagocytosis assay on isolated fixed and permeabilized RPE stained with rhodopsin, from fed or starved mice, obtained 2 h after light onset. Phagocytosis was measured as % of rhodopsin+ RPE within total RPE. n = 3 mice per condition. c, RT–PCR for Ins2 expression in RPE from fed mice at different times of the day. Lights on at 6:00 and lights off at 20:00. n = 8, 7, 7, 7, 3 and 3 mice used for times of 5:00, 6:00, 8:00, 15:00, 20:00 and 1:00, respectively. FC, fold change. d, InsR immunoprecipitated from retina lysates, at different times of day from overnight starved control and Ins2 KO mice, were probed using phospho-specific InsR antibodies. The same retina lysates were probed for GLUT4. Values were normalized to the average of control across all time points. n = 3 mice for each time point. e, Schematic of phagocytic receptors used in POS recognition (left). RT–PCR for Ins2 in RPE isolated from WT and MerTK KO or CD36 KO mice 2 h after light onset (right). n = 10 and 11 mice for WT versus MerTK KO and n = 6 and 5 mice for WT versus CD36 KO. f, Phagocytosis quantification using flow cytometry on isolated RPE from WT and MerTKCR mice (middle). n = 3 mice used per genotype. RT–PCR measuring Ins2 expression in isolated RPE from WT and MerTKCR (right). n = 14 and 10 mice for WT and MerTKCR, respectively. Plots are presented as in Fig. 1. *P ≤ 0.05,**P ≤ 0.01,***P ≤ 0.001, ****P ≤ 0.0001, one-way ANOVA (c), two-way ANOVA with Tukey’s multiple comparisons test (d), unpaired two-tailed t-test (e,f RT–PCR) and paired two-tailed t-test (f phagocytosis quantification).

    Journal: Nature metabolism

    Article Title: Phagocytosis in the retina promotes local insulin production in the eye

    doi: 10.1038/s42255-022-00728-0

    Figure Lengend Snippet: a, Representative image of three independent experiments showing immunofluorescence analysis with anti-rhodopsin (magenta) on retina sections 2 h after light onset (peak phagocytosis time) from fed and starved (10–14 h) mice. Sections were counterstained with 4,6-diamidino-2-phenylindole (DAPI) (blue). RPE is outlined in white. b, Flow cytometry-based phagocytosis assay on isolated fixed and permeabilized RPE stained with rhodopsin, from fed or starved mice, obtained 2 h after light onset. Phagocytosis was measured as % of rhodopsin+ RPE within total RPE. n = 3 mice per condition. c, RT–PCR for Ins2 expression in RPE from fed mice at different times of the day. Lights on at 6:00 and lights off at 20:00. n = 8, 7, 7, 7, 3 and 3 mice used for times of 5:00, 6:00, 8:00, 15:00, 20:00 and 1:00, respectively. FC, fold change. d, InsR immunoprecipitated from retina lysates, at different times of day from overnight starved control and Ins2 KO mice, were probed using phospho-specific InsR antibodies. The same retina lysates were probed for GLUT4. Values were normalized to the average of control across all time points. n = 3 mice for each time point. e, Schematic of phagocytic receptors used in POS recognition (left). RT–PCR for Ins2 in RPE isolated from WT and MerTK KO or CD36 KO mice 2 h after light onset (right). n = 10 and 11 mice for WT versus MerTK KO and n = 6 and 5 mice for WT versus CD36 KO. f, Phagocytosis quantification using flow cytometry on isolated RPE from WT and MerTKCR mice (middle). n = 3 mice used per genotype. RT–PCR measuring Ins2 expression in isolated RPE from WT and MerTKCR (right). n = 14 and 10 mice for WT and MerTKCR, respectively. Plots are presented as in Fig. 1. *P ≤ 0.05,**P ≤ 0.01,***P ≤ 0.001, ****P ≤ 0.0001, one-way ANOVA (c), two-way ANOVA with Tukey’s multiple comparisons test (d), unpaired two-tailed t-test (e,f RT–PCR) and paired two-tailed t-test (f phagocytosis quantification).

    Article Snippet: Antibodies used were anti-insulin (Agilent, IR002), anti-insulin (Cell Signaling, 3014), anti-C-peptide (Phoenix Pharmaceuticals, H-035-03), anti-Cre recombinase (Millipore, MAB3120), anti-rhodopsin (Abcam, ab98887), anti-cone arrestin (Millipore, AB15282), anti-phospho insulin receptor-β (Tyr1150/1151) (19H7) (Cell Signaling, 3024), anti-insulin receptor-β (Novus Biologicals, NBP2 12793), anti-Glut4 (Alomone Labs, AGT 024), anti-β actin HRP (Sigma, A3854) and anti-rabbit IgG HRP (GE Healthcare, NA934V).

    Techniques: Immunofluorescence, Flow Cytometry, Phagocytosis Assay, Isolation, Staining, Reverse Transcription Polymerase Chain Reaction, Expressing, Immunoprecipitation, Control, Two Tailed Test

    a. Quantification of OS phagocytosis by the RPE 2 hours after light onset using immunohistochemistry. Quantification of phagocytosis was measured by the amount of Rhodopsin immunoreactivity in the RPE divided by pixels and presented as phagosomes per area on the y-axis (left). Quantification of the number of phagosomes in RPE was done by counting Rhodopsin puncta in the RPE (right). n = 3 mice used for each condition. *p ≤ .05 paired two-tailed t-test. b. Western blot against Rhodopsin on isolated RPE protein lysates from fed and starved mice at 8 am and 10 am (left). Right panel is quantification of the blot to evaluate POS degradation showing 10 am band intensity as a percent of 8 am (peak phagocytosis) band intensity (right). n = 2 mice used for each time point. c. Insulin receptor was immunoprecipitated from lysates of retina from control and MerTK KO mice that were starved overnight. The lysates were probed for InsR phosphorylation or GLUT4 levels by immunoblotting. C-peptide 2 levels were determined by ELISA. N = 6 mice for each condition. *p < .05 paired two-tailed t-test. d. Schematic of WT and cleavage-resistant ‘gain of function’ MerTKCR mice with altered cleavage sites indicated (left). e. Phagocytosis quantification of ingested photoreceptor outer segments using flow cytometry on isolated RPE stained with antibody against rhodopsin from Control and Ins2 KO mice two hours after light onset. n = 7 mice used for each genotype. Plots are presented as in Fig. 1.

    Journal: Nature metabolism

    Article Title: Phagocytosis in the retina promotes local insulin production in the eye

    doi: 10.1038/s42255-022-00728-0

    Figure Lengend Snippet: a. Quantification of OS phagocytosis by the RPE 2 hours after light onset using immunohistochemistry. Quantification of phagocytosis was measured by the amount of Rhodopsin immunoreactivity in the RPE divided by pixels and presented as phagosomes per area on the y-axis (left). Quantification of the number of phagosomes in RPE was done by counting Rhodopsin puncta in the RPE (right). n = 3 mice used for each condition. *p ≤ .05 paired two-tailed t-test. b. Western blot against Rhodopsin on isolated RPE protein lysates from fed and starved mice at 8 am and 10 am (left). Right panel is quantification of the blot to evaluate POS degradation showing 10 am band intensity as a percent of 8 am (peak phagocytosis) band intensity (right). n = 2 mice used for each time point. c. Insulin receptor was immunoprecipitated from lysates of retina from control and MerTK KO mice that were starved overnight. The lysates were probed for InsR phosphorylation or GLUT4 levels by immunoblotting. C-peptide 2 levels were determined by ELISA. N = 6 mice for each condition. *p < .05 paired two-tailed t-test. d. Schematic of WT and cleavage-resistant ‘gain of function’ MerTKCR mice with altered cleavage sites indicated (left). e. Phagocytosis quantification of ingested photoreceptor outer segments using flow cytometry on isolated RPE stained with antibody against rhodopsin from Control and Ins2 KO mice two hours after light onset. n = 7 mice used for each genotype. Plots are presented as in Fig. 1.

    Article Snippet: Antibodies used were anti-insulin (Agilent, IR002), anti-insulin (Cell Signaling, 3014), anti-C-peptide (Phoenix Pharmaceuticals, H-035-03), anti-Cre recombinase (Millipore, MAB3120), anti-rhodopsin (Abcam, ab98887), anti-cone arrestin (Millipore, AB15282), anti-phospho insulin receptor-β (Tyr1150/1151) (19H7) (Cell Signaling, 3024), anti-insulin receptor-β (Novus Biologicals, NBP2 12793), anti-Glut4 (Alomone Labs, AGT 024), anti-β actin HRP (Sigma, A3854) and anti-rabbit IgG HRP (GE Healthcare, NA934V).

    Techniques: Immunohistochemistry, Two Tailed Test, Western Blot, Isolation, Immunoprecipitation, Control, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Staining

    (A-D) Immunohistochemical staining of adult mouse brain slices with antibodies against Glut4 (red), and (A, C) the presynaptic marker vGlut1, or (B, D) the Purkinje cell marker calbindin (green). Hoechst nuclear staining is shown in blue. Enlarged images of the cyan boxes in A and B show that (C) Glut4 is enriched in the synaptic-rich stratum radiatum, and (D) expressed at lower levels in Purkinje cells (arrowhead). DG, dentate gyrus; GL, granule layer; ML, molecular layer; PC: Purkinje cells; SP, stratum pyramidale; SR, stratum radiatum. CA1 and CA3 are hippocampal regions. (E-G) Immunostaining of dissociated rat hippocampal neurons with antibodies against Glut4 (red) and (E, F) the presynaptic marker synapsin, or (G) pHluorin/GFP of vGlut-pH (green) in neurons expressing Glut4 shRNA. (E) Glut4 is broadly expressed in somato-dendritic regions, (F) but also co-localizes with synapsin at nerve terminals (arrowheads) as shown in the enlarged image of the cyan box in D. (G-H) Glut4 immunofluorescence is reduced (by 70%, see STAR Methods) in neurons transfected with Glut4 shRNA and vGlut-pH, indicating the specificity of Glut4 antibody. (G) Large arrowheads point to the soma and (H) small arrowheads to boutons of the transfected neuron. Scale bars: (A, B) 50 μm, (C-H) 5 μm. See also Figure S1.

    Journal: Neuron

    Article Title: Glut4 mobilization supports energetic demands of active synapses

    doi: 10.1016/j.neuron.2016.12.020

    Figure Lengend Snippet: (A-D) Immunohistochemical staining of adult mouse brain slices with antibodies against Glut4 (red), and (A, C) the presynaptic marker vGlut1, or (B, D) the Purkinje cell marker calbindin (green). Hoechst nuclear staining is shown in blue. Enlarged images of the cyan boxes in A and B show that (C) Glut4 is enriched in the synaptic-rich stratum radiatum, and (D) expressed at lower levels in Purkinje cells (arrowhead). DG, dentate gyrus; GL, granule layer; ML, molecular layer; PC: Purkinje cells; SP, stratum pyramidale; SR, stratum radiatum. CA1 and CA3 are hippocampal regions. (E-G) Immunostaining of dissociated rat hippocampal neurons with antibodies against Glut4 (red) and (E, F) the presynaptic marker synapsin, or (G) pHluorin/GFP of vGlut-pH (green) in neurons expressing Glut4 shRNA. (E) Glut4 is broadly expressed in somato-dendritic regions, (F) but also co-localizes with synapsin at nerve terminals (arrowheads) as shown in the enlarged image of the cyan box in D. (G-H) Glut4 immunofluorescence is reduced (by 70%, see STAR Methods) in neurons transfected with Glut4 shRNA and vGlut-pH, indicating the specificity of Glut4 antibody. (G) Large arrowheads point to the soma and (H) small arrowheads to boutons of the transfected neuron. Scale bars: (A, B) 50 μm, (C-H) 5 μm. See also Figure S1.

    Article Snippet: The following primary antibodies were used: rabbit anti-Glut4 antibody (Alomone Labs AGT-024, RRID: AB_2631197), guinea pig anti-vGlut1 (Millipore AB5905, RRID: AB_2301751), mouse anti-Calbindin (SWANT C9638, RRID: AB_2314070).

    Techniques: Immunohistochemical staining, Staining, Marker, Immunostaining, Expressing, shRNA, Immunofluorescence, Transfection

    (A-D) Neurons expressing Glut4-pH were electrically stimulated with 600 AP. (A) Glut4-pH (pseudocolor) and the synaptic vesicle marker vGlut-mO (red) before and after stimulation. Neutralization of Glut4-pH vesicles with NH4Cl (white) reveals total axonal pool. (B) Average trace of Glut4-pH (n = 12 cells) with 600 AP-stimulation with the inset showing response after the first 100 AP. ΔF values were normalized to maximal ΔF from NH4Cl treatment. Error bars are shown in gray and are SEM. (C) a sample trace where stimulation was followed by quenching of extracellular pHluorin with MES acid and neutralization of Glut4-pH vesicles with NH4Cl. (D) Average surface fraction of Glut4-pH before and after stimulation (% total). Before: 7 ± 2, after 600 AP: 23 ± 4; n = 11 cells. (E-G) Glut3-pH does not mobilize at nerve terminals in response to activity. (E) Pseudocolor images of Glut3-pH in axons co-expressing the synaptic vesicle marker VAMP-mCherry (red) before and after stimulation with 600 AP (10 Hz). (F) Sample trace of Glut3-pH fluorescence (in arbitrary units) in response to stimulation. (G) Average surface fraction of Glut3-pH before and after stimulation (% toal). Before: 97 ± 1, after 600 AP: 94 ± 5; n = 5 cells. Scale bars (A, E), 5 μm. All data are shown as mean ± SEM. See also Figure S2 and S3.

    Journal: Neuron

    Article Title: Glut4 mobilization supports energetic demands of active synapses

    doi: 10.1016/j.neuron.2016.12.020

    Figure Lengend Snippet: (A-D) Neurons expressing Glut4-pH were electrically stimulated with 600 AP. (A) Glut4-pH (pseudocolor) and the synaptic vesicle marker vGlut-mO (red) before and after stimulation. Neutralization of Glut4-pH vesicles with NH4Cl (white) reveals total axonal pool. (B) Average trace of Glut4-pH (n = 12 cells) with 600 AP-stimulation with the inset showing response after the first 100 AP. ΔF values were normalized to maximal ΔF from NH4Cl treatment. Error bars are shown in gray and are SEM. (C) a sample trace where stimulation was followed by quenching of extracellular pHluorin with MES acid and neutralization of Glut4-pH vesicles with NH4Cl. (D) Average surface fraction of Glut4-pH before and after stimulation (% total). Before: 7 ± 2, after 600 AP: 23 ± 4; n = 11 cells. (E-G) Glut3-pH does not mobilize at nerve terminals in response to activity. (E) Pseudocolor images of Glut3-pH in axons co-expressing the synaptic vesicle marker VAMP-mCherry (red) before and after stimulation with 600 AP (10 Hz). (F) Sample trace of Glut3-pH fluorescence (in arbitrary units) in response to stimulation. (G) Average surface fraction of Glut3-pH before and after stimulation (% toal). Before: 97 ± 1, after 600 AP: 94 ± 5; n = 5 cells. Scale bars (A, E), 5 μm. All data are shown as mean ± SEM. See also Figure S2 and S3.

    Article Snippet: The following primary antibodies were used: rabbit anti-Glut4 antibody (Alomone Labs AGT-024, RRID: AB_2631197), guinea pig anti-vGlut1 (Millipore AB5905, RRID: AB_2301751), mouse anti-Calbindin (SWANT C9638, RRID: AB_2314070).

    Techniques: Expressing, Marker, Neutralization, Activity Assay, Fluorescence

    (A) The pH of axonal Glut4 vesicle measured from responses to acid quenching and neutralization with NH4Cl. The box and whisker plot represents median (line), 25th-75th percentile (box), and min-max (whisker). Mean pH: 6.1 ± 0.1; n = 9 cells. (B) Sample traces from boutons co-expressing Glut4-pH and vGlut-mO stimulated with 600 AP (20 Hz). (C) Decay half-time (sec) of Glut4-pH and vGlut-pH (in separate cells) after stimulation with 600 AP (10 Hz): Glut4-pH: 101 ± 17, vGlutpH: 6 ± 1; n = 7-10 cells per condition. *** P<0.001.

    Journal: Neuron

    Article Title: Glut4 mobilization supports energetic demands of active synapses

    doi: 10.1016/j.neuron.2016.12.020

    Figure Lengend Snippet: (A) The pH of axonal Glut4 vesicle measured from responses to acid quenching and neutralization with NH4Cl. The box and whisker plot represents median (line), 25th-75th percentile (box), and min-max (whisker). Mean pH: 6.1 ± 0.1; n = 9 cells. (B) Sample traces from boutons co-expressing Glut4-pH and vGlut-mO stimulated with 600 AP (20 Hz). (C) Decay half-time (sec) of Glut4-pH and vGlut-pH (in separate cells) after stimulation with 600 AP (10 Hz): Glut4-pH: 101 ± 17, vGlutpH: 6 ± 1; n = 7-10 cells per condition. *** P<0.001.

    Article Snippet: The following primary antibodies were used: rabbit anti-Glut4 antibody (Alomone Labs AGT-024, RRID: AB_2631197), guinea pig anti-vGlut1 (Millipore AB5905, RRID: AB_2301751), mouse anti-Calbindin (SWANT C9638, RRID: AB_2314070).

    Techniques: Neutralization, Whisker Assay, Expressing

    (A-D) Sample vGlut-pH traces in response to 100 AP (10 Hz) in (A) control, Glut4 KD, and Glut4 KD neurons expressing shRNA-resistant Glut4-RFP, or (B) in Glut4 KD neurons where stimulation was followed by quenching of extracellular pHluorin with MES acid. (C) Average endocytic block measured as the fraction of vGlut-pH signal remaining at two endocytic time constants (2⊺) of the control at the end of stimulation. Control: 0.15 ± 0.03, Glut4 KD: 0.7 ± 0.1, rescue: 0.26 ± 0.07; n = 13-17 cells. (D) Average exocytosis of vGlut-pH measured as ΔF at the end of 100 AP normalized to ΔFNH4Cl (% max). Control: 23 ± 4, Glut4 KD: 25 ± 4; n= 13-15 cells. All error bars are SEM. **** P<0.0001. See also Figure S4.

    Journal: Neuron

    Article Title: Glut4 mobilization supports energetic demands of active synapses

    doi: 10.1016/j.neuron.2016.12.020

    Figure Lengend Snippet: (A-D) Sample vGlut-pH traces in response to 100 AP (10 Hz) in (A) control, Glut4 KD, and Glut4 KD neurons expressing shRNA-resistant Glut4-RFP, or (B) in Glut4 KD neurons where stimulation was followed by quenching of extracellular pHluorin with MES acid. (C) Average endocytic block measured as the fraction of vGlut-pH signal remaining at two endocytic time constants (2⊺) of the control at the end of stimulation. Control: 0.15 ± 0.03, Glut4 KD: 0.7 ± 0.1, rescue: 0.26 ± 0.07; n = 13-17 cells. (D) Average exocytosis of vGlut-pH measured as ΔF at the end of 100 AP normalized to ΔFNH4Cl (% max). Control: 23 ± 4, Glut4 KD: 25 ± 4; n= 13-15 cells. All error bars are SEM. **** P<0.0001. See also Figure S4.

    Article Snippet: The following primary antibodies were used: rabbit anti-Glut4 antibody (Alomone Labs AGT-024, RRID: AB_2631197), guinea pig anti-vGlut1 (Millipore AB5905, RRID: AB_2301751), mouse anti-Calbindin (SWANT C9638, RRID: AB_2314070).

    Techniques: Expressing, shRNA, Blocking Assay

    (A-B) Glut4 mutant defective in glucose transport is recruited to synaptic surface similar to wildtype. (A) Average traces of wildtype and mutant Glut4-pH stimulated with 600 AP (10Hz). Error bars are shown in gray. (B) Average peak ΔF of Glut4-pH (% max) in response to 600 AP. Control data are the same as in Fig. 2B. Control: 17 ± 2, Glut4-pHmut: 14 ± 2; n = 6 - 16 cells. (C) Sample vGlut-pH traces in response to 100 AP (10 Hz) in control or Glut4 KD neurons expressing shRNA-resistant Glut4 mutant. All error bars are SEM.

    Journal: Neuron

    Article Title: Glut4 mobilization supports energetic demands of active synapses

    doi: 10.1016/j.neuron.2016.12.020

    Figure Lengend Snippet: (A-B) Glut4 mutant defective in glucose transport is recruited to synaptic surface similar to wildtype. (A) Average traces of wildtype and mutant Glut4-pH stimulated with 600 AP (10Hz). Error bars are shown in gray. (B) Average peak ΔF of Glut4-pH (% max) in response to 600 AP. Control data are the same as in Fig. 2B. Control: 17 ± 2, Glut4-pHmut: 14 ± 2; n = 6 - 16 cells. (C) Sample vGlut-pH traces in response to 100 AP (10 Hz) in control or Glut4 KD neurons expressing shRNA-resistant Glut4 mutant. All error bars are SEM.

    Article Snippet: The following primary antibodies were used: rabbit anti-Glut4 antibody (Alomone Labs AGT-024, RRID: AB_2631197), guinea pig anti-vGlut1 (Millipore AB5905, RRID: AB_2301751), mouse anti-Calbindin (SWANT C9638, RRID: AB_2314070).

    Techniques: Mutagenesis, Expressing, shRNA

    (A) Normalized vGlut-pH traces in response to 10, 50 and 100 AP (10Hz) before and 5 min after incubation with dGlu. (B) Normalized vGlut-pH traces of Glut4 KD neurons stimulated with 10 or 100 AP (10 Hz). (C) Average endocytic block for varying AP trains measured as the fraction of the maximal fluorescence remaining after 2 endocytic time constants in treated (dGlu or Glut4 KD) compared to control conditions, n = 4-21 cells per condition. All error bars are SEM.

    Journal: Neuron

    Article Title: Glut4 mobilization supports energetic demands of active synapses

    doi: 10.1016/j.neuron.2016.12.020

    Figure Lengend Snippet: (A) Normalized vGlut-pH traces in response to 10, 50 and 100 AP (10Hz) before and 5 min after incubation with dGlu. (B) Normalized vGlut-pH traces of Glut4 KD neurons stimulated with 10 or 100 AP (10 Hz). (C) Average endocytic block for varying AP trains measured as the fraction of the maximal fluorescence remaining after 2 endocytic time constants in treated (dGlu or Glut4 KD) compared to control conditions, n = 4-21 cells per condition. All error bars are SEM.

    Article Snippet: The following primary antibodies were used: rabbit anti-Glut4 antibody (Alomone Labs AGT-024, RRID: AB_2631197), guinea pig anti-vGlut1 (Millipore AB5905, RRID: AB_2301751), mouse anti-Calbindin (SWANT C9638, RRID: AB_2314070).

    Techniques: Incubation, Blocking Assay, Fluorescence

    (A) Representative responses of (left) Glut4-pH and vGlut-pH to treatment with 1 mM AICAR, or (right) AICAR treatment of Glut4-pH, immediately followed by MES acid quench. (B) Average traces of Glut4-pH stimulated with 300 AP (10 Hz) before and 25 minutes after incubation with 10 μM dorsomorphin, an AMPK inhibitor. (C) Average Glut4 peak ΔF (% max) in response to 300 AP before and after dorsomorphin treatment, or 600 AP with or without expression of dominant negative (DN) AMPKα1. Due to the reduction of ΔFmax values with the expression of DN AMPKα1, all ΔFmax values were normalized to the control (see Material and Methods). Control: 12 ± 1, dorsomorphin: 5 ± 1, control: 11 ± 1, DN AMPKα1: 5.9 ± 0.8; n = 6-8 cells. (D) Average Glut4-pH traces in response to 600 AP (10 Hz) in control neurons or neurons expressing TBC1D1-3A in which putative AMPK phosphorylation sites were mutated. (E) Average maximal ΔF (% max) in response to 600 AP in control and TBC1D1-3A-expressing neurons. Control data are the same as in Fig. 2B. Control: 18 ± 2, TBC1D1-3A: 8 ± 2; n = 8-12 cells. (F) Endocytosis time constant (sec) of vGlut-pH in neurons expressing TBC1D1-3A, stimulated with 600 AP (10Hz) before and after 30 minutes of dorsomorphin treatment. Before: 4.9 ± 0.7, dorsomorphin: 14 ± 2; n = 6 cells. Error bars in graphs are shown in gray (B and D). All error bars are SEM. * P< 0.05, ** P< 0.01. See also Figure S5.

    Journal: Neuron

    Article Title: Glut4 mobilization supports energetic demands of active synapses

    doi: 10.1016/j.neuron.2016.12.020

    Figure Lengend Snippet: (A) Representative responses of (left) Glut4-pH and vGlut-pH to treatment with 1 mM AICAR, or (right) AICAR treatment of Glut4-pH, immediately followed by MES acid quench. (B) Average traces of Glut4-pH stimulated with 300 AP (10 Hz) before and 25 minutes after incubation with 10 μM dorsomorphin, an AMPK inhibitor. (C) Average Glut4 peak ΔF (% max) in response to 300 AP before and after dorsomorphin treatment, or 600 AP with or without expression of dominant negative (DN) AMPKα1. Due to the reduction of ΔFmax values with the expression of DN AMPKα1, all ΔFmax values were normalized to the control (see Material and Methods). Control: 12 ± 1, dorsomorphin: 5 ± 1, control: 11 ± 1, DN AMPKα1: 5.9 ± 0.8; n = 6-8 cells. (D) Average Glut4-pH traces in response to 600 AP (10 Hz) in control neurons or neurons expressing TBC1D1-3A in which putative AMPK phosphorylation sites were mutated. (E) Average maximal ΔF (% max) in response to 600 AP in control and TBC1D1-3A-expressing neurons. Control data are the same as in Fig. 2B. Control: 18 ± 2, TBC1D1-3A: 8 ± 2; n = 8-12 cells. (F) Endocytosis time constant (sec) of vGlut-pH in neurons expressing TBC1D1-3A, stimulated with 600 AP (10Hz) before and after 30 minutes of dorsomorphin treatment. Before: 4.9 ± 0.7, dorsomorphin: 14 ± 2; n = 6 cells. Error bars in graphs are shown in gray (B and D). All error bars are SEM. * P< 0.05, ** P< 0.01. See also Figure S5.

    Article Snippet: The following primary antibodies were used: rabbit anti-Glut4 antibody (Alomone Labs AGT-024, RRID: AB_2631197), guinea pig anti-vGlut1 (Millipore AB5905, RRID: AB_2301751), mouse anti-Calbindin (SWANT C9638, RRID: AB_2314070).

    Techniques: Incubation, Expressing, Dominant Negative Mutation

    (A) Expression of tetanus toxin light chain (TeNT-LC) blocks Glut4 exocytosis membrane in response to electrical stimulation (600 AP, 10 Hz) and AICAR treatment. (B) Munc13 KD does not block AICAR-driven Glut4 exocytosis while it only partially inhibits activity-driven exocytosis (600 AP, 10 Hz). (C) Average peak ΔF (% max) in response to 600 AP or AICAR in the same genotypes as shown in A and B. 600 AP (control: 20 ± 3, TeNT-LC: 1 ± 1, Munc13 KD: 6 ± 2); AICAR (control: 20 ± 3, TeNT-LC: 0 ± 2, Munc13 KD: 17 ± 4); n = 3-13 cells. Error bars in graphs are shown in gray (A and B). All error bars are SEM. See also Figure S6.

    Journal: Neuron

    Article Title: Glut4 mobilization supports energetic demands of active synapses

    doi: 10.1016/j.neuron.2016.12.020

    Figure Lengend Snippet: (A) Expression of tetanus toxin light chain (TeNT-LC) blocks Glut4 exocytosis membrane in response to electrical stimulation (600 AP, 10 Hz) and AICAR treatment. (B) Munc13 KD does not block AICAR-driven Glut4 exocytosis while it only partially inhibits activity-driven exocytosis (600 AP, 10 Hz). (C) Average peak ΔF (% max) in response to 600 AP or AICAR in the same genotypes as shown in A and B. 600 AP (control: 20 ± 3, TeNT-LC: 1 ± 1, Munc13 KD: 6 ± 2); AICAR (control: 20 ± 3, TeNT-LC: 0 ± 2, Munc13 KD: 17 ± 4); n = 3-13 cells. Error bars in graphs are shown in gray (A and B). All error bars are SEM. See also Figure S6.

    Article Snippet: The following primary antibodies were used: rabbit anti-Glut4 antibody (Alomone Labs AGT-024, RRID: AB_2631197), guinea pig anti-vGlut1 (Millipore AB5905, RRID: AB_2301751), mouse anti-Calbindin (SWANT C9638, RRID: AB_2314070).

    Techniques: Expressing, Blocking Assay, Activity Assay