FIG. 4.

FoxO3a is required for SirT1-induction of antioxidant genes. (A, B) BAEC were first infected o/n with Ad-SirT1 or a control adenovirus, 36 h later cells were infected o/n with Ad-shFoxO3a or a control adenovirus and finally serum deprived for 8 h. Expression levels of ROS detoxification genes were determined by qRT-PCR (A) and Western Blot (B). (*) p≤0.05 versus control. (C, D) BAEC whole cell extracts were subjected to immunoprecipitation (IP) with a specific FoxO3a (C) or anti-HA (D) antibody or normal rabbit IgG, as a control, and analyzed by western blot as indicated. Input indicates a 1/10 of whole cell extract not subjected to immunoprecipitation. (C) BAEC were treated with 10 mM nicotinamide for 15 h (D) BAEC were coinfected o/n with Ad-SirT1 and Ad-FoxO3a-WT. 24 h postinfection the cells were serum deprived o/n, and then treated for 4 h with 50 μM H₂O₂ or vehicle (E) Confluent BAEC were grown in galactose media in the absence of serum for 48 h. Whole cell extracts were subjected to immunoprecipitation (IP) with a specific FoxO3a or SirT1 antibody or normal IgG, as a control, and analyzed by western blot as indicated. Data are from ≥3 independent experiments. Data are means+SD (*) p≤0.05 versus control. The blots presented correspond to a representative experiment.