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. 2020 Sep 30;37(2):145–165. doi: 10.1007/s12264-020-00581-9

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© The Author(s) 2020

Open AccessThis 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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 3 — Specificity of the novel antibody against the hASIC1a ectodomain. A Verification of antibody specificity in neurons by surface staining with ASC06-IgG1 in Asic1a^−/− neurons transfected with GFP-hASIC1a. Fluorescent signal of surface hASIC1a occurs only in the transfected neuron. Scale bar, 50 μm. B, C Species specificity of ASC06-IgG1 against ASIC1a tested by transfecting CHO cells with GFP-tagged hASIC1a, rASIC1a, or mASIC1a and surface staining with ASC06-IgG1. B Representative images of surface staining (red) and total ASIC1a expression (green) (scale bar, 20 μm). C Quantification of surface labeling based on ASC06-IgG1/GFP fluorescence intensity ratios. Data are presented as the mean ± SEM. [hASIC1a, 0.44 ± 0.02 (n = 11 cells); rASIC1a, 0.20 ± 0.02 (n = 9 cells); mASIC1a, 0.12 ± 0.01 (n = 13 cells); **P < 0.01, ***P < 0.001, ****P < 0.0001, one-way ANOVA multiple comparison]. D, E Acid-activated current of GFP-hASIC1a, -rASIC1a, or -mASIC1a expressed in CHO cells. Cells were transfected with the same amount of cDNA for the three orthologs followed by whole-cell recording of I_6.0. D Representative I_6.0 traces. E Quantification of current density of peak I_6.0 (pA/pF). Data are presented as the mean ± SEM. [hASIC1a, 378.47 ± 52.22 (n = 6 cells); rASIC1a, 197.73 ± 31.30 (n = 8 cells); mASIC1a, 157.98 ± 30.15 (n = 8 cells), **P < 0.01, no significant difference between rASIC1a and mASIC1a, one-way ANOVA multiple comparison]. F Endogenous rASIC1a detected by ASC06-IgG1 in primary cultures of rat cortical neurons immunostained at DIV 18 with ASC06-IgG1 (green) under non-permeabilized conditions; neurons were then stained with the neuronal marker NeuN (red), astrocyte marker GFAP (magenta), and nuclear maker DAPI (blue) (scale bar, 50 μm).