S-Nitrosation of Cellular Proteins by NO Donors in Rat Embryonic Fibroblast 3Y1 Cells: Factors Affecting S-Nitrosation
Figure 1
Protein S-nitrosation induced by potential S-nitrosating agents in 3Y1 cell lysates. (a) Detection of S-nitrosated proteins in cell lysate treated with potential S-nitrosating agents. 3Y1 cell lysates prepared in PBS containing 0.5 mM EDTA and 0.5% Triton X-100 were incubated with CysNO (200 μM), GSNO (200 μM), NOR-3 (200 μM), or SIN-1 (1 mM) at 37°C for 30 min. S-nitrosated proteins were detected by the biotin-switch assay. (b) Specificity of the biotin switch assay. 3Y1 cells lysates were incubated with or without GSNO (500 μM), and the resulting S-nitrosated proteins were assessed by biotin-switch assay as above, but with or without ascorbate in the biotinylation step. A 10% gel was used for this confirmation experiment. Therefore, the resolution of S-nitrosated protein bands was different from that in all other experiments in which 6.0%–16.5% gradient gels were used.
