2.2. Assessment of Nrf2 Nuclear Expression

HMECs were seeded at a density of 1,000,000 per well and grown in 6-well plates until they reached full confluence, then cells were incubated with bardoxolone methyl, dimethyl fumarate, and L-sulforaphane for three hours. To measure Nrf2 expression in the nucleus, a Nuclear Extract Kit (Active Motif) was used to isolate the nuclear and cytoplasmic fractions. Nuclear and cytoplasmic extracts (25 or 30 μg protein/sample) were processed as follows: first, the membranes were scanned for total protein content for further band normalization, and subsequently, they were incubated with primary rabbit polyclonal Anti-Nrf2 Antibody (H-300) (sc-13032, Santa Cruz Biotechnology, lot No. GR197455-1) 1 : 1000 overnight and with goat anti-rabbit secondary antibody (sc-2004, Santa Cruz Biotechnology, lot No. 12314) 1 : 2500 for 45 minutes. After chemiluminescent band detection, membranes were washed and incubated with primary Anti-Lamin A/C Monoclonal Antibody (mab636, Thermo Fisher Scientific, lot No. QF215120) 1 : 1000 overnight and with anti-mouse secondary antibody (sc-516102, Santa Cruz Biotechnology) 1 : 5000 for 1 hour. Bands were again detected with the use of chemiluminescence, measured and normalized to total protein content using Image Lab Software (Bio-Rad).