Casein based buffer

For the next phase, 20 µg of protein cell extracts were separated using a reducing 12% SDS–PAGE and transferred onto PVDF membrane. The membranes were blocked with casein-based buffer (Sigma-Aldrich, St. Louis, MO, USA), incubated with primary antibodies to iNOS and RAGE proteins, and then probed with secondary antibodies raised against the appropriate species. Loading of identical amounts of protein fractions on each line was confirmed using antibodies against GAPDH protein and by Ponceau S staining of the blots. Proteins were detected using ECL chemiluminescence. The blots were scanned and quantified densitometrically. The relative protein abundances of iNOS and RAGE proteins were normalized to the level of GAPDH protein changes in expression were assessed with a heteroscedastic two-tailed t-test.

Cell lysates (total and enriched SNO fractions) were separated using 12% SDS-PAGE and transferred to PVDF membranes (0.22 μm). The membranes were blocked with casein-based buffer (Sigma-Aldrich, St. Louis, MO, USA), incubated with primary antibodies specific for a protein of interest followed by secondary HRP-conjugated antibodies. Protein bands were detected using the ECL chemiluminescence system (Amersham, London, UK). GelQuant software (Version 2.7, Neve Yamin, Israel) compared densitometrically the amounts of S-nitrosylated proteins under all studied conditions. A heteroscedastic two-tailed t-test was used to assess the changes in S-nitrosylation statistically.