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. 2020 Jul 7;10:11188. doi: 10.1038/s41598-020-67702-2

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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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Cumulative effects of repetitive EA treatments on the expression of PAR-2, CGRP and c-Fos in the spinal cord of the goats. (a) Western blotting of PAR-2, CGRP and c-Fos in the spinal cord of the goats. All electrophoresis was performed under the same condition. The immuno-detected protein bands, appeared on a horseradish peroxidase substrate (Millipore, Billerica, MA, USA), were visualized using the ImageQuant LAS 4,000 min CCD camera (GE Healthcare, Piscataway Township, NJ, USA). The bands were analysed using a Quantity One software (Bio-Rad), and the result is expressed as the immunoreactivity ratio of the target gene to β-actin. Insets show a representative band of the proteins expressed in all groups. Full-length blots are presented in Supplementary Fig. S1. Data were normalized against β-actin and expressed as the mean ± SD, n = 6. Semi-quantitative analysis of (b) PAR-2 expressions; P = 0.000, VH/Sham-A group versus Control group; P = 0.000, VH group versus Sham-A group; P = 0.000, VH group versus EA group [F (3, 20) = 1,040.60, P = 0.000]. (c) CGRP expressions; P = 0.000, VH/Sham-A group versus Control group; P = 0.000, VH group versus Sham A group; P = 0.000, VH group versus EA group [F (3, 20) = 1819.47, P = 0.000]. (d) c-Fos expressions; P = 0.000, Sham-A or VH or EA group versus Control group; P = .000, VH group versus Sham-A group; P = 0.000, EA group versus VH group [F (3, 20) = 223.53, P = 0.000]. **P < 0.01, VH/Sham-A/EA group versus Control group; ⁺⁺P < 0.01, VH/EA group versus Sham-A group; ^##P < 0.01, VH group versus EA group. One-way ANOVA followed by Bonferroni’s post-hoc test.