FIG. 4.
GSNO increases S-nitrosylation of STAT3. (A). The effects of GSNO (2 h treatment) on the S-nitrosylation of STAT3 or its upstream kinase JAK2 and receptor gp130 in BV2 cells were examined by biotin switch method. For analysis of total S-nitrosylated proteins, Western analysis for biotin-labeled proteins was performed (A-i). For analysis of S-nitrosylation of STAT3, JAK2, and receptor gp130, the biotinylated proteins were pulled down with avidin–agarose conjugate and the levels of STAT3, JAK2, and gp130 were determined by Western analysis (A-ii). (B) The effect of GSNO (0.5 mM for 2 h) and LPS (0.1 μg/ml for 12 h) treatment on S-nitrosylation of cellular proteins (B-i) and STAT3 (B-ii) was also examined in primary cultured rat microglia. (C) The effects of GSNO (500 μM for 2 h) on IL-6-induced (30 ng/mL for 0.5 h) phosphorylation of STAT3 (Tyr705) and JAK2 (Tyr1007/1008) were examined by the Western analysis (C-i). The phosphorylation of gp130 was examined by immunoprecipitation of phospho-Tyr containing proteins and following Western analysis of gp130 (C-ii). The levels of β-actin were used as internal loading control. (D) The effect of GSNO (2 h) on S-glutathionylation of STAT3 and other cellular proteins in BV2 cells were analyzed by immunoprecipitation and Western analysis. For positive control, the control cell lysates were incubated with oxidized and reduced glutathione mixture (GSSG/GSH; 5 mM each; pH 7.0) for 1 h at room temperature in the presence or absence of 20 mM dithiothreitol (DTT). (E) STAT3 S-nitrosylation (E-i) and S-glutathionylation (E-ii) in BV2 cells treated with GSNO (0.5 mM for 2 h) were further analyzed by sandwich ELISA using antibodies specific to S-nitrosocysteine or GSNO and STAT3. The vertical lines in E indicate the standard error mean; **p>0.01; not significant (N.S.)>0.05 compared with vehicle (VHC) treated group. ELISA, enzyme-linked immunosorbent assay; JAK2, Janus-activated kinase 2.