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. 2020 Jan 10;11:174. doi: 10.1038/s41467-019-13889-6

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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. 4 — a–c Representative pictures and quantification (d) of western blot analysis of LRS, SESN1, and SESN2 protein levels in TA muscles from WT (white bars) and PHD1^mKO male and female (blue bars) mice. e PHD1^KO myogenic progenitors were transduced with lentiviruses to over-express myc-LRS (LRS^OE), an empty vector (EV) was used as control. Representative pictures (left panel) and quantification (right panel) of western blot analysis of S6K1 phosphorylation and myc-LRS expression levels in differentiated WT (white bars), PHD1^KO (red bars), PHD1^KO + EV (light gray bars) and PHD1^KO + LRS^OE myotubes (dark gray bars) after 1 h starvation (starved) or 30 min stimulation with 5 mM leucine (leucine). f Representative picture (left panel) and quantification (right panel) of western blot analysis of RagA leucylation (K_Leu142) levels in WT (white bars) and PHD1^KO (red bars) myotubes after 1 h starvation (starved) or 30 min stimulation with different doses of leucine (leucine). g Representative picture (left panel) and quantification (right panel) of western blot analysis of RagA leucylation (K_Leu142) levels in TA from WT (white bars) and PHD1^KO (red bars) female mice. Representative pictures (h) and quantification (i) of colocalization between mTOR (red) and LAMP2 (green) in WT (white bars) and PHD1^KO (red bars) myotubes. Merged (gray) picture shows the outline of the colocalization analysis between mTOR and LAMP2 that was performed to generate data shown in panel (i). Dots represent quantification of individual myotubes from three independent experiments. Statistics: two-way ANOVA test with a Holm–Sidak post hoc test (e, i) or unpaired t test (d, f, g) (*p < 0.05; **p < 0.01; ***p < 0.001; ns not significant). Each dot represents a single mouse. Dots represent experimental duplicates from four independent experiments (e, f). Bar graphs represent mean ± SEM (error bars). Data is presented as fold change to WT starved (e, f) or fold change to WT (d, g). See also Supplementary Fig. 4. Source data are provided as a Source Data file.