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. 2018 Dec 3;138(23):2682–2697. doi: 10.1161/CIRCULATIONAHA.118.034541

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© 2018 The Authors.

Circulation is published on behalf of the American Heart Association, Inc., by Wolters Kluwer Health, Inc. This is an open access article under the terms of the Creative Commons Attribution Non-Commercial-NoDerivs License, which permits use, distribution, and reproduction in any medium, provided that the original work is properly cited, the use is noncommercial, and no modifications or adaptations are made.

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Figure 7. — The p.Q1283H variant affects the integration of RyR2 with PP2Ac likely because of decreased AnkB binding of B56α. A, Representative immunoblots of the expression levels of relevant phosphatases (PP1c and PP2Ac) from left ventricle sections in littermates of WT and KI mice. B, Protein phosphatase assays using a fluorescence-based kit for measuring the total PP1 and PP2A activities in heart homogenates from WT and KI mice. C and D, Representative immunoblots (C) and quantitative assessment (D) of PP1c and PP2Ac coimmunoprecipitated with RyR2 showing a reduced amount of PP2Ac associated with RyR2 (**P<0.01), but the interaction of PP1c with RyR2 was not affected in KI hearts. E, Representative immunofluorescence assays of WT (upper) and KI (lower) cardiomyocytes coimmunolabeled with PP2Ac (left, green) and RyR2 (middle, red) specific antibodies. F, Representative coimmunoprecipitation assays showing a macromolecular complex composed of AnkB, B56α, PP2Ac, and RyR2. Total protein homogenates from mouse left ventricles were immunoblotted with either an anti-RyR2 antibody or with antibodies that recognize AnkB, B56α, and PP2Ac, all of which were detected in immunoprecipitates, whereas control preimmune serum (IgG) did not immunoprecipitate the proteins. G and H, Representative coimmunoprecipitation assays (G) and quantitative assessment (H) using the anti-B56α antibody as bait showing decreased binding of RyR2 (**P<0.01) or AnkB (**P<0.01) with B56α, but the abundance of PP2Aα or PP2Ac binding with B56α was unchanged in KI hearts. I, Coimmunoprecipitation from HEK293 cells expressing GFP-tagged mutant AnkB and mCherry-tagged B56α showing a reduced binding of AnkB to B56α. J, Representative GST pull-down assays showing that GST-labeled WT AnkB pulled down recombinant B56α, but GST-labeled mutant AnkB showed a decreased binding to B56α. K, BIAcore-binding properties of WT (K1: K_D=1.64±0.05 μM) or mutant (K2: K_D=22.17±6.42 μM) AnkB-purified proteins to B56α. AnkB indicates ankyrin-B; HEK, human embryonic kidney; GAPDH, glyceraldehyde-phosphate dehydrogenase; IB, immunoblots; IP, immunoprecipitation; ISO, isoproterenol; KI, knockin; KI, knockin; PP1c, PP1 catalytic subunit; PP2Aα, PP2A scaffolding subunit; PP2Ac, PP2A catalytic subunit; RU, response unit; RyR2, ryanodine receptor type 2; and WT, wild type.