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. 2021 Oct 8;12:5913. doi: 10.1038/s41467-021-25944-2

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© The Author(s) 2021

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. 1 — a Representative images of the back skin and tail of 7-week old OTULIN^fl/fl and Δ^KerOTULIN mice. b Representative images of H&E-stained skin sections of 7-week old mice of the indicated genotypes. Scale bar: 100 μm. The two lower panels show verrucous carcinomas in 11-week old Δ^KerOTULIN mice. Boxed area depicts magnified area shown below. Scale bar for magnified areas: 200 μm. NL non-lesional; L lesional; VC verrucous carcinoma. c Epidermal thickness quantification at 7 weeks of age (n = 5 per condition; ****p < 0.0001, One-way ANOVA with multiple comparisons). Data represent means ± SEM. d Trans-epidermal water loss (TEWL) measurements at week 7, 8 and 9 of age (n = 5 mice per condition; **p = 0.0019; ****p < 0.0001, One-way ANOVA with multiple comparisons). Data represent means ± SEM. e Immunofluorescent staining of skin sections from 7-week old OTULIN^fl/fl mice and non-lesional (NL) and lesional (L) skin of Δ^KerOTULIN mice with antibodies against CD11b (red) and F4/80 (green) (upper panel), filaggrin (middle panel) or keratin-6 (lower panel) and nuclear staining with Dapi. Each staining was performed on n = 5 mice per condition. Scale bar: 25 μm. f Relative mRNA expression of IL-4, IL-6, IL-13, TNF, S100A8, and MCP-1 in epidermal tail lysates of 8-weeks-old OTULIN^fl/fl (WT; n = 8 and n = 5 for MCP-1 analysis) and Δ^KerOTULIN (KO; n = 8) mice. Data represent means ± SEM. (***p < 0.001; Mann−Whitney two-sided testing). g Levels of IL-6, TNF, IL-17, and MCP-1 in serum of 8-weeks-old OTULIN^fl/fl (WT; n ≥ 7) and Δ^KerOTULIN (KO; n = 8) mice (**p < 0.01; ***p < 0.001; ****p < 0.0001; Mann−Whitney two-sided testing was performed between relevant genotypes). h Western blotting on epidermal tail lysates of 7-week old OTULIN^fl/fl (n = 3) and Δ^KerOTULIN (n = 3) mice using antibodies detecting OTULIN, linear ubiquitin chains (M1-Ubq), IκBα, and phospho-IκBα. Immunoblotting for phospho-IκBα and consecutively for IκBα was performed on the same blot. Molecular weight marker units are in kilodalton (kD). Anti-actin is shown as loading control. The representative image is shown for three independent experiments.