Methods to analyze S-nitrosylation of proteins involved in apoptosis

Nitric oxide (NO) is an important signaling molecule that plays a key role in various physiological and pathological processes. One of the well-established mechanisms by which NO regulates the function of various target proteins is through S-nitrosylation. NO readily reacts with thiol (SH) groups in the cysteine residues of target proteins to form nitrosothiol (S-NO) groups. This posttranslational modification of proteins can positively or negatively regulate various signaling pathways including apoptosis. Likewise, S-nitrosylation of various apoptosis-regulatory proteins has been demonstrated to modify the apoptotic response to various stimuli. We have shown that NO nitrosylates important antiapoptotic proteins, such as Bcl-2 and FLIP, and prevents their downregulation via the ubiquitin-proteasomal degradation pathway. To detect protein S-nitrosylation, we isolated the protein by immunoprecipitation and analyzed cysteine nitrosylation by Western blotting or spectrofluorometry.