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. 2020 Mar 13;11:1381. doi: 10.1038/s41467-020-14895-9

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 6 — Muscles from fed and fasted (2 days) mice are analyzed. In fasting, contralateral limbs were transfected. Data are mean ± SEM. P values by one-tailed t test. Fluorescence intensity was obtained using the Imaris software. a Top: Muscle cross-sections stained with indicated antibodies. Bar, 20 μm. n = four independent experiments. Bottom: Fluorescence intensity. n = 4. *P < 0.05 vs. fed. b Equal membrane extracts from muscles were analyzed by immunoblotting. n = two independent experiments. c Left: Plakoglobin immunoprecipitation from muscle membranes from mice treated with colchicine or vehicle was analyzed by immunoblotting. n = two independent experiments. Right: Densitometric measurement of presented blots. d Left: Muscle cross-sections from fasted mice treated with colchicine or vehicle were stained with indicated antibodies. Bar, 20 μm. n = three independent experiments. Right: Fluorescence intensity. n = 3. *P < 0.05 vs. vehicle injection. e Membranes purified from muscles expressing shLacz or 6His-plakoglobin were analyzed by glycerol gradients and immunoblot. Red rectangle marks fractions containing glycosylated-β-dystroglycan in shLacz-expressing muscles. n = four independent experiments. f Illustration of two potential LIR motifs in β-dystroglycan using iLIR database. g Left: In vitro competition assay: plakoglobin immunoprecipitation from membrane extracts from muscles of fed mice, followed by incubation in vitro with synthetic peptides corresponding to β-dystroglycan’s-LIR domains or scrambled control. β-Dystroglycan LIR-2 peptide efficiently competed with β-dystroglycan on binding to plakoglobin. Right: Densitometric measurement, β-dystroglycan to plakoglobin ratio in protein precipitates following incubation with synthetic peptides. n = four independent experiments. *P < 0.05 vs. scrambled. h Left: Cross-sections of muscles expressing GFP-β-dystroglycan or GFP-β-dystroglycan-ΔLIR from fasted mice were stained with indicated antibodies. Bar, 20 μm. n = three independent experiments. Right: Fluorescence intensity of areas of potential colocalization on the plasma membrane. n = 3. *P < 0.05 vs. GFP-β-dystroglycan. Bottom: Colocalization of indicated proteins in region of interest (region of co-occurrence, data are ratio to GFP-β-dystroglycan), and the corresponding Pearson’s correlation coefficients of colocalization. n = 7, two independent experiments. *P < 0.05 and **P < 0.005 vs. GFP-β-dystroglycan. i Proposed model: Under normal conditions, basal recycling of plakoglobin-DGC-insulin receptor co-assemblies occurs via plakoglobin masking β-dystroglycan LIR domains. During fasting, when autophagy is induced, LC3 competes with plakoglobin on binding to β-dystroglycan’s LIR domains, directing the formed vesicles to the lysosome.

Fig. 6 — Muscles from fed and fasted (2 days) mice are analyzed. In fasting, contralateral limbs were transfected. Data are mean ± SEM. P values by one-tailed t test. Fluorescence intensity was obtained using the Imaris software. a Top: Muscle cross-sections stained with indicated antibodies. Bar, 20 μm. n = four independent experiments. Bottom: Fluorescence intensity. n = 4. *P < 0.05 vs. fed. b Equal membrane extracts from muscles were analyzed by immunoblotting. n = two independent experiments. c Left: Plakoglobin immunoprecipitation from muscle membranes from mice treated with colchicine or vehicle was analyzed by immunoblotting. n = two independent experiments. Right: Densitometric measurement of presented blots. d Left: Muscle cross-sections from fasted mice treated with colchicine or vehicle were stained with indicated antibodies. Bar, 20 μm. n = three independent experiments. Right: Fluorescence intensity. n = 3. *P < 0.05 vs. vehicle injection. e Membranes purified from muscles expressing shLacz or 6His-plakoglobin were analyzed by glycerol gradients and immunoblot. Red rectangle marks fractions containing glycosylated-β-dystroglycan in shLacz-expressing muscles. n = four independent experiments. f Illustration of two potential LIR motifs in β-dystroglycan using iLIR database. g Left: In vitro competition assay: plakoglobin immunoprecipitation from membrane extracts from muscles of fed mice, followed by incubation in vitro with synthetic peptides corresponding to β-dystroglycan’s-LIR domains or scrambled control. β-Dystroglycan LIR-2 peptide efficiently competed with β-dystroglycan on binding to plakoglobin. Right: Densitometric measurement, β-dystroglycan to plakoglobin ratio in protein precipitates following incubation with synthetic peptides. n = four independent experiments. *P < 0.05 vs. scrambled. h Left: Cross-sections of muscles expressing GFP-β-dystroglycan or GFP-β-dystroglycan-ΔLIR from fasted mice were stained with indicated antibodies. Bar, 20 μm. n = three independent experiments. Right: Fluorescence intensity of areas of potential colocalization on the plasma membrane. n = 3. *P < 0.05 vs. GFP-β-dystroglycan. Bottom: Colocalization of indicated proteins in region of interest (region of co-occurrence, data are ratio to GFP-β-dystroglycan), and the corresponding Pearson’s correlation coefficients of colocalization. n = 7, two independent experiments. *P < 0.05 and **P < 0.005 vs. GFP-β-dystroglycan. i Proposed model: Under normal conditions, basal recycling of plakoglobin-DGC-insulin receptor co-assemblies occurs via plakoglobin masking β-dystroglycan LIR domains. During fasting, when autophagy is induced, LC3 competes with plakoglobin on binding to β-dystroglycan’s LIR domains, directing the formed vesicles to the lysosome.