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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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AC9 association with Hsp20 is Yotiao independent. (A) Heart extracts from WT or AC9^−/− mice were subjected to IP with IgG (control) or anti-Hsp20. Hsp20-associated AC activity was stimulated with 300 nM Gαs (n = 4; ^#P < 0.001 IgG versus Hsp20; **P < 0.01 WT versus AC9^−/−). (B,C) HEK293 cells were transfected with Flag-tagged AC9, myc-tagged Yotiao, and V5-tagged Hsp20 as indicated. Cell lysates were immunoprecipitated with anti-Flag (B) or anti-Hsp20 (C). AC9-Hsp20 protein complexes were detected by WB (n = 3). (D) Proximity ligation assay for Hsp20 and YFP-tagged AC9. HEK293 cells were transfected with the indicated plasmids. Representative images are shown. PLA signals were quantified by high content microscopy (positive cells defined as 4 signals or “dots” per cell; ~2000 cells imaged per condition). *P < 0.05 t-test, n = 3. Full-length WBs are presented in Supplementary Fig. S6.