Figure 2.

S-Nitrosoglutathione and S-nitroso-N-acetylcysteine are more active than NO, GSH, or GSSG in affecting 5-LO expression; the effect does not involve glutathionylation or oxidation, and it is both transcriptional and translational. (A) Western blot analysis was performed on whole cell extracts. Lane 1, control; lanes 2 and 3, GSNO at 1 and 10 μM; lanes 4 and 5, GSH at 1 and 10 μM; lanes 6 and 7, GSSG at 1 and 10 μM; lanes 8 and 9, (NO at 1 and 10 μM, prepared anaerobically from a saturated solution in deaerated water); lanes 10 and 11, S-nitroso-N-acetyl-cysteine (SNOAC) at 1 and 10 μM. The membrane was stripped and re-probed with α-tubulin to verify equal amount protein loading. Twenty-five micrograms of protein was loaded in each lane for each experiment (n = 3). (B) Blots were scanned and densitometry was performed for quantification. (C) Western blot analysis was performed on whole cell extracts made from A549 cells, treated with 15 or 25 μg/ml actinomycin D beginning 2 h before 6 h exposure to 1 μM GSNO—or in the absence of GSNO. Cells were also treated with 50 or 75 μg/ml cycloheximide for 15 min before and during a 6-h exposure to 1 μM GSNO and throughout the 6-h GSNO exposure. A, actinomycin D; C, cycloheximide. Twenty five micrograms of protein was loaded in each lane for each experiment (n = 3; P < 0.005). (D) Blots were scanned and densitometry was performed for quantification.