2.5. Biotin Switch assay

Protein S-nitrosation levels were determined in macrophage lysates using the Biotin Switch assay with some modifications from the original protocol [16]. All steps before the neutravidin-purification of biotinylated proteins were carried out in the dark. After treatments, macrophages were collected and lysed by scraping in TENT buffer (50 mmol/l Tris-HCl pH 7.2, 150 mmol/l NaCl, 1 mmol/l EDTA, 0.1 mmol/l neocuproine, 1% Triton X-100, protease inhibitor cocktail) and incubation on ice for 15 min. The soluble fraction was obtained by centrifugation at 10000 g and 4 °C for 15 min. Protein concentration was determined using the Bradford protein assay (Bio-Rad) and samples were adjusted to 0.5 mg/ml with TEN buffer (TENT without Triton X-100). Free thiols were blocked in 4 volumes of HENS buffer (250 mmol/l Hepes pH 7.7, 1 mmol/l EDTA, 0.1 mmol/l neocuproine, 1% SDS) containing 20 mmol/l S-methyl methanethiosulfonate at 50 °C for 30 min with agitation every 5 min. Proteins were precipitated with cold acetone for at least 30 min at −20 °C to remove excess blocking reagent. To reduce SNO-thiols and label resulting SH-residues, precipitated proteins were resuspended in 300 µl HENS/mg protein containing 5 mmol/l ascorbate and incubated in the presence of 1 mmol/l (N-[6-(Biotinamido)hexyl]-3′-(2′-pyridyldithio)propionamide (HPDP-biotin, Thermo Fisher Scientific) for 2 h at room temperature. After aceton precipitation, biotinylated proteins were resuspended in 150 µl HENS/mg protein and purified by incubation with NeutrAvidin Plus Ultra Link Resin (Thermo Fisher Scientific) in 5 volumes neutralization buffer (20 mmol/l Hepes pH 7.7, 100 mmol/l NaCl, 1 mmol/l EDTA, 0.5% Triton X-100) over night at 4 °C. Bound proteins were washed extensively (20 mmol/l Hepes pH 7.7, 600 mmol NaCl, 1 mmol/l EDTA, 0.5% Triton X-100), eluted from the resin at 37 °C for 20 min with 1 mmol/l β-mercaptoethanol in 20 mmol/l Hepes pH 7.7, 100 mmol/l NaCl, 1 mmol/l EDTA, and analyzed using Western Blot.