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. 2017 Jul 17;7:5522. doi: 10.1038/s41598-017-05816-w

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© The Author(s) 2017

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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Design and verification of the AC9^−/− mouse model. (A) Schematic of AC9 gene-trap strategy and genotyping primers. Intron distances are not drawn to scale. (B) PCR analysis of genotyping: lanes 1, 2, and 3 represent wild-type (WT), AC9^+/− (Het) and AC9^−/−, respectively. (C) AC9 protein levels were detected by immunoprecipitation of pre-immune (PI) or Yotiao complexes from WT and AC9 KO heart extracts followed by western blotting (WB) with anti-AC9 antibody. AC9 protein is not detectable in total heart extracts by WB (n = 5). (D) Real time PCR of cardiac AC isoforms in AC9^−/− heart, normalized to WT expression levels (n = 3; mice 1 month of age). Loss of AC9 mRNA was confirmed with primer sets 9-1 and 9-2. Full-length WBs are presented in Supplementary Fig. S6.