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. 2017 Aug 30;7:9938. doi: 10.1038/s41598-017-09275-1

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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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Effect of HNO-mediated PKGIα oxidation on kinase activity. HEK-293 cells were transfected to express PKGIα WT or various mutants Cys42Ser, Cys117/195Ser, Cys42/117/195Ser, exposed to (A) NCA (100 µmol/L, 30 min) or (B) AS (500 µmol/L, 15 min). In vitro kinase (IVK) assays were performed in cell lysates by addition of recombinant His₆-tagged C1-M-C2 domain of cardiac myosin-binding protein C as a substrate in the presence of γ³²P-ATP. Substrate phosphorylation was detected by autoradiography (figure shows cropped version, full representation in online supplement). Bar charts represent the results of 5 independent experiments. Data are expressed in comparison to the WT response after HNO treatment. *P < 0.05, **P < 0.01, ***P < 0.001 by comparison against each respective unstimulated control or WT after HNO-exposure. Western immunoblot analysis for PKGIα under non-reducing (NR) or reducing (R) conditions was performed in the same samples (figure shows cropped reducing blots, full representation in online supplement). Representative immunoblots show PKGIα migrating at 75 kDa (monomer) and 150 kDa (dimer). Data are representative of 5 independent experiments. (C) Analysis of HNO-induced oxidation of PKGIα. HEK-293 cells were transfected to express PKGIα WT or various mutants Cys42Ser, Cys117Ser, Cys195Ser, Cys117/195Ser, Cys42/117/195Ser, exposed to NCA (100 µmol/L, 30 min, upper panel) or the respective controls namely DMSO or decomposed NCA donor compound or AS (500 µmol/L, 15 min, bottom panel) or the respective controls NaOH, nitrite (NO₂ ⁻) or decomposed AS. Western immunoblot analyses for PKGIα under non-reducing (NR) or reducing (R) conditions were performed. Representative immunoblots show PKGIα migrating at 75 kDa (monomer) and 150 kDa (dimer) under non-reducing and at 75 kDa under reducing conditions. Data are representative of 5 independent experiments. n.s.: non-significant; dec: decomposed.