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. 2023 Jan 26;24(3):439–451. doi: 10.1038/s41590-022-01418-5

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© The Author(s) 2023

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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Extended Data Fig. 4 — (a) Schematic representation showing that the initial targeting construct (upper; knock-out first construct) for the Snx25 gene was transformed to an Snx25^fl/fl (for exon 4) construct after recombination by flippase (FLP). (b) VF thresholds of WT, Snx25^+/−, and Snx25^fl/fl mice are shown (WT: n = 6; Snx25^+/−: n = 7; Snx25^fl/fl: n = 5). Snx25^+/+/Snx25^+/−, p = 0.004; Snx25^+/+/Snx25^fl/fl, p = 0.001. g, gram. (c) Formalin responses in the phase 1 (left, 0–10 min, Snx25^+/+/Snx25^+/−, p = 0.014) and phase 2 (right, 20–60 min, Snx25^+/+/Snx25^+/−, p = 0.011) for three lines of mice (WT: n = 6; Snx25^+/−: n = 7; Snx25^fl/fl: n = 7). s, second. (d) Experimental paradigm and schedule. The Avil Cre driver functions specifically in DRG neurons. TAM feeding for two weeks was employed to differentiate control (No TAM) and experimental (TAM) groups. Another control was Snx25^fl/fl mice without Cre driver and with TAM feeding. (e) Representative immunoblotting showed the expression levels of SNX25 in the DRG (L4) of Snx25^Avil-cKO mice in the presence or absence of TAM. (f) VF thresholds are plotted for Snx25^fl/fl and Snx25^Avil-cKO mice treated with TAM (Snx25^fl/fl: n = 19; Snx25^Avil-cKO: n = 24, p = 0.068). g, gram. (g) VF thresholds were plotted for Snx25^Avil-cKO mice before and after TAM treatment (n = 12, p = 0.165). g, gram. (h) Formalin responses of the two groups of mice (Snx25^fl/fl : n = 5, Snx25^Avil-cKO: n = 11). The responses in the phase 1 (left, 0–10 min, p = 0.529) and phase 2 (right, 20–60 min, p = 0.747) are indicated. s, second. (i) Expression profiles of mRNAs for pain-related factors (Trpv1, Scn9a, Scn10a) in DRG (L4) of Snx25^fl/fl and Snx25^Avil-cKO are shown (Snx25^fl/fl: n = 6; Snx25^Avil-cKO: n = 6). Trpv1, p = 0.687; Scn9a, p = 0.422; Scn10a, p = 0.576. Results are represented as mean ± SEM. Statistical analyses were performed using the two-tailed Student’s t-test (f–i) or one-way ANOVA (b and c), and significant differences between group means were identified with the Tukey–Kramer test. *p < 0.05, **p < 0.01. n.s., not significant.

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