Figure 6.

Nox4-dependent changes in Akt phosphorylation in cultured cardiac cells. (A) Protein levels of Nox4, actin as loading control, phospho (Ser⁴⁷³)-Akt (p-Akt), and total Akt were assessed by western blotting in lysates from H9C2 cells after siRNA-mediated knockdown of Nox4 (siNox4) or adenoviral-mediated Nox4 overexpression (Nox4). Scrambled siRNA (Scr) and overexpression of β-Gal were used as respective controls. Cells were cultured in 10% FCS containing medium. (B) Effects of PEG-catalase treatment (CAT 250 U/mL) or vehicle control (CAT 0 U/mL) on p-Akt levels following Nox4 (+Nox4) or control β-Gal overexpression (-Nox4). (C and D) Assessment of p-Akt and total Akt protein levels after stimulation with IGF-I (10 ng/mL) in Nox4 knockdown cells (+siNox4) or controls (-siNox4: scrambled siRNA was used) (C), and in cells with Nox4 overexpression (+Nox4) vs. control (-Nox4: transduction with a β-Gal expressing adenovirus) (D). Immunoblots were quantified by densitometry; data are the mean of 3–4 independent experiments. **P < 0.01 using unpaired Student’s t-test (A, C, D) and one-way ANOVA followed by Bonferroni post-hoc test for multiple comparisons (B) with # indicating P < 0.01 vs. control condition (-Nox4, 0 U/mL PEG-catalase).