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. 2020 Oct 19;11:5267. doi: 10.1038/s41467-020-18942-3

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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. 7 — a, b We assayed if Stub1 depletion affected peroxisome quality using NIH3T3 cells expressing a peroxisomal redox sensor roGFP2-PTS1. a roGFP2-PTS1 emission ratios between 405 and 488 nm excitation. Each dot represents one peroxisome, and a column denotes all peroxisomes within a single cell. The ratios in Stub1-depleted cells vs. those in control cells are shown (top: 48 h after depletion; bottom: 96 h after depletion). b Graph tabulating the percentage of peroxisomes with roGFP2-PTS1 ratios between 1.5–2 (green) and ≧2 (red). c, d The effect of WT and Ataxia-related Stub1 mutant overexpression on ubiquitin and p62 recruitment onto ROS-stressed peroxisomes in Stub1 KO (NIH3T3) cells. Stub1 KO cells expressed EGFP-Ub (c) or EGFP-p62 (d), plus TagBFP-PMP34, diKillerRed-PTS1, and WT hStub1 or Ataxia-related Stub1 mutants tagged with TagBFP. c WT, n = 11 cells; N65S, n = 14 cells; A79D, n = 9 cells; L123V, n = 8 cells; K144X, n = 9 cells; M240T, n = 11 cells; T246M, n = 6 cells. Each mutant vs. WT Stub1: N65S, P = 0.0123; A79D, P = 0.0315; L123V, P = 0.0143; K144X, P = 0.0066; M240T, P = 0.0119; T246M, P = 0.0023 (one-tailed t test). d WT, n = 13 cells; N65S, n = 10 cells; A79D, n = 13 cells; L123V, n = 10 cells; K144X, n = 11 cells; M240T, n = 9 cells; T246M, n = 12 cells. Each mutant vs. WT Stub1: N65S, P = 0.0259; A79D, P = 0.0186; L123V, P = 0.0021; K144X, P = 0.0038; M240T, P = 0.0090; T246M, P = 0.0100 (one-tailed t test). e The effect of Ataxia-related Stub1 mutant expression on LC3B recruitment onto ROS-stressed peroxisomes in NIH3T3 cells expressing diKillerRed-PTS1, EGFP-LC3B, and PMP34-TagBFP. Control, n = 10 cells; L123V, n = 10 cells; K144X, n = 13 cells; M240T, n = 11 cells; T246M, n = 11 cells; H261Q, n = 10 cells. Each mutant vs. Control: L123V, P = 0.0016; K144X, P = 0.0023; M240T, P = 0.0012; T246M, P = 0.0012; H261Q, P = 0.0013 (one-tailed t test). All error bars in c–e represent the mean ± SEM. Source data are provided as a Source data file.