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Nitrosative stress was quantified using slot blotting. Briefly, 30 μg of proteins of each sample were loaded in each cell of the slot blot apparatus. Vacuum was applied to transfer the proteins to the nitrocellulose paper. The membranes were blocked using 5% non-fat milk in TBST (1% tween 20 in tris buffered saline) for 1 hour. Membranes were probed with anti-Nitro-tyrosine antibody (Cayman chemical; Ann Arbor, MI) Nitro-tyrosine levels were quantified by measuring the optic density of the bands using image J software.
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