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. 2022 Jul 9;13:3984. doi: 10.1038/s41467-022-31762-x

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© The Author(s) 2022

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. 5 — Physical interactions of mouse (a) human (b) NEMEP with GLUT1 and GLUT3. Lysates from HEK293T cells co-transfected with plasmids encoding GLUT1-FLAG or GLUT3-FLAG or control vector and NEMEP-HA (as indicated) were immunoprecipitated with anti-FLAG affinity beads, and immune complexes were analyzed by immunoblotting using an antibody against HA. The protein inputs were detected with western blotting using indicated antibodies. c Immunoprecipitation of endogenous NEMEP from NEMEP-3XFLAG knock-in mESCs using anti-FLAG antibody. The immune complexes were analyzed by immunoblotting using antibodies against FLAG and GLUT1 and GLUT3. The protein inputs were detected with western blotting using indicated antibodies. d Lysates from HEK293T cells co-transfected with plasmids encoding mGLUT1-FLAG or mGLUT3-FLAG and NEMEP (GFP-tagged, wild type or indicated mutant variants) immunoprecipitated with anti-GFP-trap affinity beads; immune complexes were analyzed by immunoblotting using an antibody against FLAG. The protein inputs were detected with western blotting using indicated antibodies. Glucose consumption (e) and lactate excretion (f) in WT and CRISPR-dCas9-VP64 (CRISPRa) mediated NEMEP overexpressing mESCs. Data are means ± S.E.M., n = 3 biological independent samples. g WT and CRISPR-dCas9-VP64 (CRISPRa) mediated NEMEP overexpressing mESCs were supplied with 25 mM glucose, 2 µM of oligomycin (ATP synthase inhibitor), and 50 mM 2-DG (a glucose analog that inhibit glycolysis) at the indicated time. ECAR was examined using a Seahorse XFe96 analyzer. The values are normalized to the protein concentration (means ± S.E.M., n = 8 biological independent samples). h Glucose uptake analysis in mESCs cells stably expressing control plasmid or plasmids with WT or mutant variants of NEMEP, as indicated. The values are normalized to the protein concentration (means ± S.E.M., n = 3 biological independent samples). i,j Glucose uptake analysis in HEK293T cells expressing plasmids for overexpression of the indicated proteins or protein pairs. The values are normalized to the protein concentration (means ± S.E.M., n = 3 biological independent samples). a–j: Data are the representative of three independent experiments with similar results. P values were determined by unpaired two-tailed t-test (e, f), one-way ANOVA (h) with Sidak’s corrections or two-way ANOVA (i, j) with Tukey’s corrections. Source data are provided as a Source Data file.