Figure 2.

Mini-GAGR increases the nuclear translocation and transcription factor activity of Nrf2. A, mouse cortical neurons (E17, DIV11–14) were treated with 1 μm mini-GAGR for 0, 1, 3, 6, and 24 h and processed for immunocytochemistry with antibodies against Nrf2 (green; Alexa Fluor 488), βIII tubulin (red; Alexa Fluor 594), and DAPI (blue) (scale bar, 30 μm). B, the specificity of anti-Nrf2 antibody (80 kDa) was confirmed by immunoblotting of the cytosol of untreated mouse cortical neurons. C, the fluorescence intensities of nuclear Nrf2 in images were quantified using Metamorph to calculate the average intensity ± S.E. (error bars) of nuclear Nrf2 staining during the time course and used for bar graphs: compared with control, 3 h (p < 0.001, n = 30 cells) and 6 h (p = 0.058, n = 30 cells). (One-way ANOVA (F(4, 145) = 6.2641, p < 0.001) and Bonferroni's multiple-comparison test were used). D, the transcription factor activity of Nrf2 was measured 3 h after mini-GAGR treatment via absorbance at 450 nm for the bar graphs: control (0.094 ± 0.002, n = 8 different batches of embryos) and mini-GAGR (0.124 ± 0.005, n = 8 different embryo batches) (p < 0.001) (***, p < 0.001; unpaired t test, two-tailed).