Nrf2 Activation Assay in Lens Epithelial Cells

Nrf2 activation assay was carried out as described in the company’s protocol (TransAM Nrf2 Transcription Factor Assay Kit, Cat No. 50296, Active motif, Carlsland, CA, USA) and as described in our published protocol [11 (link),67 (link)]. Briefly, 10 µg of nuclear extract (up to 10 µL diluted with complete lysis buffer) prepared from H₂O₂ exposed LECs was added to the strips well following the addition of 40 µL binding buffer containing 20 pmol of the wild-type or mutated consensus oligonucleotide to each well. The plate was incubated for 1 h at room temperature (RT). Then, 100 µL primary antibody (1:1000 dilution) was added after 3 washes and then incubated at RT for 1 h. 100 µL of diluted anti-rabbit HRP conjugated antibody (1:1000 dilution) was added and incubated for 1 h at RT. Then, 100 µL of developing solution was added to wells after washing and incubated for 2 to 10 min in the dark. Finally, 100 µL of stop solution was added, and Optical Density (O.D.) was measured at 450 nm.