Detection of NRF2 nuclear translocation by immunofluorescence

Cells were seeded in 8-chamber culture slides. On the second day, cells were treated with ABS (pH4, 200 μM) for 20 min, followed by replacement with regular culture medium for 2 h. Cells were fixed with 4% paraformaldehyde in PBS at room temperature for 45 min, followed by permeabilization with 0.5% Triton X-100 in PBS for 2 min on ice. After blocking, cells were incubated with primary antibody against NRF2 (ABE413, Millipore Sigma, Burlington, MA) overnight at 4° C. Cells were incubated with anti-rabbit secondary antibody labelled with Fluor-488 for 1 h at room temperature. After washing, cells were covered with Vectashield mounting medium with DAPI (Vector Laboratories, Burlingame, CA).