5-HT induced ERK1/2 phosphorylation diverges from the transactivation pathway at or after NADPH oxidase.

<p>(A) SH-SY5Y cells were treated with 0.01 to 100 µM H₂O₂ for 5 min. Following drug treatments, cell lysates were evaluated by Western blot analysis as described in Materials and Methods. Data were normalized to total ERK1/2 protein expression and are expressed as the fold change (average ± S.E.M.) in phospho-ERK immunoreactivity compared to vehicle-treated cells. (B) SH-SY5Y cell cultures were pretreated with vehicle or 10, 100 or 1000 µM of the ROS scavenger <i>N</i>-acetyl-l-cysteine (NAC) for 45 min followed by treatment with vehicle or 100 nM 5-HT for 5 min and lysates were evaluated as in “A”. Cell cultures were also pretreated with vehicle or the NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI) (C) or apocynin (D) for 45 min followed by treatment with vehicle or 100 nM 5-HT for 5 min, and results were analyzed for phospho-ERK1/2 as described in “A”. (E) Cultures were pretreated with vehicle or 0.1 µM of the PKC inhibitor Go 6983 for 45 min followed by treatment with vehicle or 100 nM 5-HT for 5 min, and results were analyzed for phospho-ERK1/2 as described above. Representative blots of phospho-ERK1/2 and total ERK1/2 at 42 and 44 kDa are shown. (Data are representative of 4-8 independent experiments. * = p < 0.05 compared to vehicle-treated cells; # = p < 0.05 compared to 5-HT-treated cells, one-way ANOVA, Tukey post-test).