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. 2021 Mar 2;12:1379. doi: 10.1038/s41467-021-21711-5

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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 N4BP1 domain structure and positions of ENU-induced mutations (red lines). b Relative concentration of TNF in the culture medium of peritoneal macrophages harvested from mice of the stash (blue), winter (red), and acorn (green) pedigrees 4 h after stimulation with 20 ng/ml of R848. REF, N4bp1^+/+; HET, N4bp1^+/mutation; and VAR, N4bp1^{mutation/mutation}, where “mutation” represents the stash, winter, or acorn allele. c Manhattan plot showing p values of association between the phenotype of elevated TNF production in response to R848 and mutations identified in the three pedigrees in b calculated using a recessive model of inheritance. The −log10 p values were plotted versus the chromosomal positions of mutations. Horizontal lines indicate thresholds of p = 0.05 with (red) or without (purple) the Bonferroni correction. The p value for linkage of N4bp1 mutations with the elevated TNF production is indicated. d Immunoblot analysis of N4BP1 and GAPDH in N4bp1^+/+, N4bp1^+/ac and N4bp1^ac/ac peritoneal macrophages. e–h TNF concentration in the culture medium of N4bp1^+/+, N4bp1^+/ac and N4bp1^ac/ac peritoneal macrophages treated with Pam3CSK4, *P = 0.0275, *P = 0.0481, *P = 0.0175, *P = 0.0213, two-way ANOVA and post hoc Tukey test (e), R848, *P = 0.0372, *P = 0.0154, ***P = 0.0004, *P = 0.0148, two-way ANOVA and post hoc Tukey test (f), poly(I:C) (g), and LPS (h). n = 4 mice per genotype. i–l TNF concentration in the culture medium of N4bp1^+/+, N4bp1^+/−, and N4bp1^−/− peritoneal macrophages treated with 40 ng/ml Pam3CSK4, ***P = 0.0006 (i), 20 ng/ml R848, ****P < 0.0001 (j), poly(I:C) (k), and LPS (l). n.s. not significant. One-way ANOVA and post hoc Tukey test (i–l). m Immunoblot analysis of N4BP1 and GAPDH in N4bp1^+/+, N4bp1^+/−, and N4bp1^−/− peritoneal macrophages. Each symbol (b, i–l) represents an individual mouse. Data are representative of two (d–h, m) or three (i–l) independent experiments (mean ± s.d. in b, e–l). P values are listed in order from left to right. Source data are provided in the Source Data file.