S-Nitrosation of Cellular Proteins by NO Donors in Rat Embryonic Fibroblast 3Y1 Cells: Factors Affecting S-Nitrosation
Figure 3
Effects of DETAPAC, DTNB, and L-leucine on CysNO-induced S-nitrosation in 3Y1 cells. (a) NO release from S-nitrosothiols in HBSS. CysNO or GSNO was diluted in HBSS to a final concentration of 200 μM without or with DETAPAC (0.5 mM). NO concentration in the medium was aerobically monitored at 37°C using an NO electrode. The trace shown is a representative result. (b and c) Effects of DETAPAC, DTNB, and L-leucine on CysNO-induced S-nitrosation in 3Y1 cells. Cells in HBSS were treated with CysNO (200 μM) for 30 min in the absence or presence of DETAPAC (0.5 mM), L-leucine (1 mM), and DTNB (100 μM), which were added 20 min before the addition of CysNO. Protein S-nitrosation was detected using (b) the DAN assay and (c) the biotin-switch assay. The blot shown is a representative result. (d) The efficiency of CysNO- and D-CysNO-induced S-nitrosation, each at 200 μM, was measured by the DAN assay as in (b). S-nitrosothiol levels in cells treated with CysNO alone (labeled “none” in (b)) were taken as 100% (12 ± 2 nmol SNO/mg protein), and the values are expressed as the mean ± SEM of six to eight independent experiments in (b) and from three independent experiments in (d). *.
