Figure 4

Effect of EGCG on LPS-induced protein S-nitrosylation in BV-2 cells. (A) Dose titration for EGCG. BV-2 cells were treated with 0, 5, 10, and 20 μM EGCG for 20 hours and cell viability was assessed by a MTT assay (# P <0.01, control vs. 20 μM EGCG, n = 3). (B) Administration of EGCG (10 μM) 1 hour prior to LPS (100 ng/mL) exposure inhibited NO production in BV-2 cells (# P <0.01, LPS vs. LPS + EGCG, n = 3). (C) A total of 59 differentially S-nitrosylated protein spots were detected by NitroDIGE analysis (fold change > 1.3, P <0.05, LPS vs. LPS + EGCG, n = 3). (D) Seventy-eight proteins were identified from the above spots by LC-MS/MS, and SOD2 (a), PRDX (b), and USP14(c) were selected for validation by the BST method. After Biotin-HPDP labeling and biotin affinity pull-down, individual protein Western blotting was performed. The amount of SNO-proteins was quantified by a densitometer, normalized to total proteins, and expressed as percentage of untreated controls. Data are means ± SEM (n = 3); * P <0.05, untreated vs. LPS; # P <0.05, LPS vs. LPS + EGCG; ** P <0.05, untreated vs. EGCG. The results showed the down-regulation of S-nitrosylation levels of these proteins in “LPS + EGCG” compared to LPS-treated samples.