2.8. Nrf2 nuclear translocalization and activity assay

HK-2 cells were plated on coverslips, exposed to HG with or without mitoQ treatment, and stained with anti-Nrf2 and secondary antibodies. Cell nuclei were stained with DAPI, and Nrf2 translocation was observed using confocal microscopy. [27] Nrf2-antioxidant response element (ARE) binding was measured using a TransAM Nrf2 Kit (active motif), as previously described. [29] Briefly, 10 μg of nuclear protein was incubated in a 96-well plate and coated with oligonucleotides containing a consensus binding site for Nrf2. The plate was incubated with an anti-Nrf2 antibody and HRP-conjugated secondary antibody. The absorbance was measured at 450 nm and reflected Nrf2 activity. [29].