Fig. 6. H₂O₂-mediated oxidative stress involves MAPK/NF-κB dependent signaling. PBMC were treated with H₂O₂ for induction of oxidative stress or with vehicle (mock). Samples were collected at 15 min, 30 min, 2h, and 4h time intervals following H₂O₂-treatment and extracted proteins were analyzed by western blotting as described in materials and methods to detect phosphorylated vs. total ERK1/2, c-Jun, p38, and NF-κB. Incubation was not extended up to 10h since optimal phosphorylation times of these signaling proteins are limited. (A) Western blots depict the expression of phosphorylated and total signaling proteins at 15 min, 30 min, 2h, and 4h intervals following H₂O₂ treatment. Densitometry data (mean±SEM) were used to calculate phosphorylated to total protein ratios. H₂O₂ induced the optimal phosphorylation of (B) ERK1/2 (17.32±0.17 fold), (C) c-Jun (4.20±0.05 fold), (D) p38 (14.13±0.19 fold), and (E) NF-κB (2.10±0.01 fold) at 30 min, 15 min, 2h, and 30 min, respectively, as compared to mock (P<0.0001).