FIGURE 1.

Localization of FOXO3a-like immunoreactive signal in Hdh cells and validation of FOXO3a antibody. A, representative micrographs demonstrating differential distribution of FOXO3a antibody staining in Hdh^7/7, Hdh^7/109, and Hdh^109/109 cells. B, immunocytochemical analysis with FOXO3a antibodies in Hdh^7/7 cells left untreated or treated with 1 mm 3-NP for 48 h. C, co-localization of EGFP signal and FOXO3a antibody staining in HEK293 cells transfected with EGFP-FOXO3a encoding construct. In A–C, DNA was counterstained with DAPI. Scale bar, 20 μm. D, Western blot analysis with FOXO3a antibodies of HEK293 cells left untransfected (control) or transfected with Flag-FOXO3a encoding construct, scrambled siRNA, or siRNAs 1 and 2 against FOXO3a. Flag-specific antibodies were used for verification of Flag-FOXO3a expression and tubulin β served as loading control. E, quantification of the data in D. FOXO3a signals were normalized to tubulin β signals. F, RT-qPCR analysis of FOXO3a mRNA levels in siRNAs transfected HEK293 cells performed in parallel with Western blotting. In E and F, the mean results from two independent experiments are shown.