Biotin nem

S-nitrosated proteome from cell extract was purified using a modified “biotin-switch” assay (13 (link),17 (link),18 (link)). After corresponding treatments on HEKn cells, the following steps were performed in the dark. Briefly, cells were harvested in RIPA cell lysis buffer (Thermo Scientific), sonicated, and centrifuged at 13,500 rpm for 15 min at 4 °C to remove cellular debris. 10 mM N-ethylmaleimide (NEM) was added and incubated for 1 h at room temperature to block all free Cys residues. Excess NEM in samples was removed by ice-cold acetone precipitation repeated three times for 20 min each. 1 mM ascorbic acid (Vc, Sigma-Aldrich) and 500 μM biotin-NEM (Thermo Scientific Pierce) were then added and incubated at room temperature for 1 h in order to reduce and label S-nitrosated Cys residues. Labeled S-nitrosated proteome was purified using streptavidin agarose beads (Thermo Scientific Pierce). Then the samples were resolved by electrophoresis through 10% SDS-polyacrylamide gels. Proteins were transferred to nitrocellulose membranes (Bio-Rad) and probed with PARP antibody (Cell Signaling Inc. #9532). The membranes were developed using the SuperSignal chemiluminescent detection system (Thermo Scientific Pierce). Quantification of immunoblotting results was performed using software ImageJ. Statistical summaries were obtained from 3 independent samples.