Sta 305

Purified human HDL (MyBioSource, # MBS173147, total protein 40 mg/ml, standard curve range 1–12 ng/ml) was used as standard for the determination of plasma concentrations of apoAI. For NT-apoAI standard, purified human apoAI (Biomedical Technologies, # BT-927) was nitrated with sodium nitrite ^{23 (link), 24 (link)}. Briefly, purified apoAI (~ 1 mg/ml) was adjusted to pH 3.5 and mixed with 200 mM sodium nitrite and then incubated at 37°C for 24 h under low agitation. Samples were dialyzed against phosphate buffer saline (PBS) for 24 h at 4°C, and protein concentration was determined by the Coomassie Reagent (Thermo Scientific, #23236) and bovine serum albumin (BSA) as a standard ^{25 (link)}. The amounts of nitrated tyrosines in the modified apoAI were determined using a commercially available NTyr ELISA kit based on nitrated albumin (Cell Biolabs, # STA-305) as described by the manufacturer. The nitrated apoAI (0.7 mg/ml protein) contained 2000 nM NT-apoAI giving a concentration of 80 mmol of nitrated tyrosine per mol of apoAI.