Peroxynitrite

Ammonium bicarbonate ( $\ge$ 98%, Ph. Eur., BP, Carl Roth, Karlsruhe, Germany) was dissolved in pure water to yield a final buffer concentration of 2 M, and the pH was adjusted to 7.8 by the addition of 1 M hydrochloric acid (37% stock solution, Merck Millipore, Darmstadt, Germany). For each reaction, 300 or $500\mu$ L of protein solution was transferred into a brown reaction tube (Eppendorf, Hamburg, Germany), and 7.7 or $12.8\mu$ L ammonium bicarbonate buffer (2 M) was added to yield a final buffer concentration of 50 mM. After being thawed on ice, sodium peroxynitrite (160–200 mM, Merck Millipore) was added to the protein solutions. To yield molar ratios of ONOO^– over tyrosine residues (ONOO^–/Tyr) of 1/1, 3/1, or 5/1, 0.6, 1.8, or $3\mu$ L ONOO^– were added to $300\mu$ L samples of Bet v 1 and Phl p 5, $4.9\mu$ L ONOO^– to $300\mu$ L samples of OVA (5/1), and 1, 3, or $5\mu$ L ONOO^– to $500\mu$ L samples of Bet v 1 and Phl p 5. The reaction was performed on ice for 110 min. Afterwards, the sample was pipetted into a 10 kDa centrifugal filter (Amicon®, Merck Millipore) and centrifuged at 14 000 $\times$ g for 2 min (5427 R, Eppendorf). The sample was washed five times with $200\mu$ L PBS and centrifugation at 14 000 $\times$ g for 2 min. For sample recovery, the filter was turned upside down, transferred into a clean microcentrifuge tube, and centrifuged at 1 000 $\times$ g for 2 min. To recover possible sample residues, the filter was washed with $200\mu$ L pure water and centrifuged upside down at 1 000 $\times$ g for 2 min into the concentrated protein sample. Two to six independent protein samples were prepared for the different ONOO^–/Tyr ratios. For mock controls of the allergens (termed mock-treated Bet v 1 and mock-treated Phl p 5 hereafter), protein solutions were treated with all buffers but without ONOO^–, and were purified as described above.

For each examined individual experiment, a fresh Dihydrorhodamine (DHR)-123 (Sigma-Aldrich, Chemie GmbH, Taufkirchen, Germany) dye working solution was prepared. DHR-123 is a fluorescence probe commonly used for measuring ROS [56 (link)]. It can passively diffuse through cell membranes and then, after being exposed to intracellular nitric oxide and peroxynitrite, DHR-123 is oxidized to rhodamine-123, which exhibits green fluorescence and stains mitochondria inside a living cell [57 (link)], thus allowing detection by flow cytometer. After isolating eosinophil subtypes, flow cytometer test tubes (Corning Falcon, Newport, Tennessee, USA) were prepared (5 × 10⁴ of an eosinophil subtype in each tube), and 1x PBS was added to a final volume of 200 μL. A separate test tube with 200 μL DHR-123 was prepared to evaluate reagent contamination. Each experimental tube was supplemented with DHR-123 working solution (final concentration 750 ng/mL), mixed by gentle vortexing, and incubated for 45 min, a sufficient amount of time for fluorescence evaluation [58 (link)], at standard conditions (5% CO₂ in air at 37 °C). For flow cytometry calibration, a test tube with 5 × 10⁴ eosinophils in PBS, but without DHR-123, was prepared. After incubation, the relative amount of ROS formed was evaluated in a flow cytometer by measuring the mean fluorescence intensity (MFI) of the investigated eosinophil subtype population.

In situ PLA was used for evaluation of FKBP12/RyR stability in human myotubes under nitro-oxidative stress, as previously described [19 (link)]. Briefly, immortalized human myoblasts (Myology Institute, Paris, France) were differentiated into myotubes and nitro-oxidative stress was induced by addition of the peroxynitrite donor 3-morpholinosydnonimine (SIN1, 5 mM, SCBT, Heidelberg, Germany) for 30 min. Myotubes were fixed with 4% paraformaldehyde (Aname, Madrid, Spain) and incubated overnight with the following primary antibodies: anti-RyR mouse mAb (1:200, Thermo Scientific, Waltham, MA, USA), and anti-FKBP12 rabbit pAb (1:100, Novus Biologicals, Centennial, CO, USA). The Duolink in situ PLA Orange assay kit was used with corresponding conjugated secondary antibodies (Sigma). Samples were counterstained with FITC-conjugated Myosin Heavy Chain-CFS mAb (MyHC, 1:50, R&D, Minneapolis, MN, USA) and mounted with ProLong Gold antifade reagent (Life Technologies, Eugene, OR, USA). Image quantification was performed with ImageJ (NIH). For each image, the total number of spots was normalized to the MyHC area. At least 3 images per condition were analysed with an average of 8–9 myotubes per image. Statistical significance was determined using One-Way ANOVA followed by Dunett’s multiple comparisons test. p-values < 0.05 were considered statistically significant.