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. 2019 Jan 10;10:115. doi: 10.1038/s41467-018-07918-z

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© The Author(s) 2019

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. 3 — Two distinct ANO1 isoforms are expressed in red and white skeletal muscle of zebrafish. a Likelihood-based phylogenetic tree (Treefinder, version 2011) from aligned amino acid sequences of mouse (m) ANO1 (GenBank accession no. NP_848757), zebrafish (zf) isoforms ANO1-a (accession no. XP_009301746) and ANO1-b (accession no. XP_017209649), mouse ANO2 (accession no. NP_705817), zebrafish isoforms ANO2-a (accession no. XP_009298253) and ANO2-b (accession no. XM_009293432), with the ancestral Drosophila ANO channel (accession no. NP_648535) as outgroup, shows species-independent isoform clustering. Likelihood score, −16,906.7; edge lengths optimised. Scale bar, evolutionary distance in substitutions per site (s/s). b RT-PCR amplification from first strands of whole adult zebrafish with primers specific for all 4 ANO channel isoforms (Supplementary Table 1) showed signals with similar intensity (P > 0.05, n = 5) for RNAs that were isolated from adult normal zebrafish. c RT-PCR amplification with ANO2-specific primers under identical PCR conditions did not show any signal, neither from red muscle (r.m.) nor white muscle (w.m.). d In contrast, ANO1-specific primers amplified PCR fragments from both muscle types but to different extents. e Similarly, control PCR amplifications with DHPα_1S-specific primers detected both, α_1S-a and α_1S-b in both tissues but again to different extents. f Left graph, quantification of band intensities of ANO1 isoforms revealed that ANO1-a is predominantly expressed in the red muscle compared (P < 0.001) to white muscle (43.75 ± 1.89%, n = 5), and ANO1-b is mainly expressed in the white muscle compared (P < 0.001) to red muscle (45.56 ± 2.08%, n = 5). Right graph, DHPRα_1S-a and α_1S-b, known to be exclusively expressed in red and white muscles, respectively⁴, displayed a similar cross-contamination pattern for DHPRα_1S-a in white (24.21 ± 3.12%; n = 5) and DHPRα_1S-b in red muscle (49.90 ± 8.09%; n = 5). Bars represent mean ± s.e.m. and overlaying blue stars indicate individual data points; *** P < 0.001 determined by unpaired Student’s t-test