Detection of S-Nitrosylated Proteins in Cardiac Tissue

Hearts were rapidly excised, perfused and equilibrated and homogenized as above. S-nitrosylated proteins were detected by the biotin-switch method as previously described [31 (link)]. First, free thiols (-SH) in tissue homogenates were blocked for 1 h at 50°C in the dark with methyl methanethiosulfonate (MMTS). Proteins were precipitated with 4 volumes of ice-cold acetone, washed repeatedly with acetone to remove free MMTS and resolubilized. Thereafter, nitrosylated cysteine residues (-SNO) were reduced to free cysteine by incubating 1 h with 30 mM sodium ascorbate and selectively labeled with HPDP-biotin. Proteins were precipitated again with acetone to wash the excess of HPDP-biotin and solubilized with HENS buffer (HEN buffer with 1% SDS). Following solubilization, the samples were incubated 1 h with agarose-conjugated streptavidin beads (Sigma-Aldrich Chemical, St Louis, MO, USA) and centrifuged to pull HPDP-biotinylated proteins down. Adsorbed proteins were resolved in a 10% SDS-PAGE, electroblotted to a nitrocellulose membrane and stained with Ponceau red to evaluate the total protein content. Then, the biotinylated proteins were probed with anti-Cx43 (Sigma Aldrich Chemical, St Louis, MO, USA) antibodies. For all Western blot analysis, the intensity of the signal was evaluated using the ImageJ program (NIH public domain software).