Figure 1. BARD robustly activates Nrf2 signaling cascade in HUVECs. Human umbilical vein endothelial cells (HUVECs) were treated with Bardoxolone Methyl (BARD, at 10-100 nM) and cultured for applied time periods, Nrf2-Keap1 binding was tested by a co-immunoprecipitation assay (A); Expression of listed protein in cytosol fraction lysates (B, G) and nuclear fraction lysates (C) was tested by Western blotting, with expression of listed Nrf2 pathway mRNAs examined by qPCR (E, F); The relatively ARE (antioxidant response element) activity was also tested (D). Expression of the listed proteins was quantified, normalizing to the indicated loading control protein. (A–C, G) Error bars stand for mean ± standard deviation (SD, n=5). “Veh” stands for vehicle control (same for all Figures). ** p<0.01 vs. “Veh” (D, E) Each experiment was repeated five times to insure the consistency of experimental results.