FIG 4.

Nox5α siRNA reduces both phosphorylation of Erk and AKT and ROS production. (A) Lysates were prepared from MT2 cells transfected with scrambled siRNA (SC) or a Nox5-specific siRNA (siNox5α or siNox5α-I) and were subjected to immunoblotting with anti-Nox5 or anti-β-actin antibodies. (B) MT1 and MT2 cell lines stably transfected with Nox5α siRNA (MT1siNox5α and MT2siNox5α) or scrambled siRNA (MT1SC and MT2SC) were established. Expression levels of Nox5α mRNAs were examined by real-time PCR using GAPDH (glyceraldehyde-3-phosphate dehydrogenase) as an internal control. The data represent means ± SD (n = 3) of results from three separate experiments. Student's t test was performed. (C) Expression levels of endogenous Nox5α proteins in the indicated cell lines were determined by immunoblotting with anti-Nox5 antibodies.β-Actin was used as a loading control. Alternatively, phosphorylation levels of Erk and AKT were examined by immunoblotting with anti-phospho-Erk and anti-phospho-AKT antibodies. (D) Levels of intracellular ROS in MT1siNox5α, MT1SC, MT2siNox5α, and MT2SC cells were measured by luminol assay in the presence or absence of catalase (250 U/ml). The data represent means ± SD (n = 4) of results from three separate experiments. Statistical analysis was performed with ANOVA, followed by the Bonferroni test.