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.
Azad
Neelam
N
Department of Pharmaceutical Sciences, West Virginia University, Morgantown, WV, USA.
Iyer
Anand Krishnan V
AK
Rojanasakul
Yon
Y
D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western / Q000379:methods*; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods*; D009569:Nitric Oxide / Q000032:analysis / Q000378:metabolism*; D011506:Proteins / Q000032:analysis* / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000032:analysis / Q000378:metabolism
D011506:Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D009569:Nitric Oxide