Anti nrf2 antibody

HCEC (1 × 10⁵ cells) were seeded on coverslips in 6-well plates. After 24 h, HCEC were treated with increasing doses of hemin (0–100 µM) and incubated for 8 h. Immunofluorescence staining was performed as reported^{65 (link)}. Upon fixation with methanol at −20 °C for 10 min, cells were washed twice with PBS and then incubated for 1 h with blocking solution consisting of 5% bovine serum albumin (BSA) in PBS with 0.3% Triton X-100. The samples were incubated with an anti-Nrf2 antibody (Genetex; diluted 1:500 in PBS/0.2% Triton X-100) or an anti-HO1 antibody (Genetex; diluted 1:1,000 in PBS/0.2% Triton X-100) overnight at 4 °C. After several washing steps, the samples were incubated with an appropriate Alexa488-coupled goat-anti-rabbit secondary antibody (Life Technologies; 1:400 in PBS plus 0.2% Triton X-100). Nuclei were finally counterstained using TO-PRO-3 (Life Technologies; 1:100 in PBS). Coverslips were mounted using VectaShield (Vector Labs, Burlingame, USA) and analyzed with a Zeiss Axio Observer.Z1 microscope equipped with a confocal LSM710 laser-scanning unit (Zeiss, Oberkochen, Germany). Pictures were analyzed and processed using Image J.

Cells (5 × 10⁶) were cross-linked followed by preparation of nuclear lysates using Magna ChIP^TM protein G Kit (Millipore, Burlington, MA, USA). Nuclear lysates were sonicated to shear DNA to around 500 bp followed by immunoprecipitation for 16 h at 4 °C using IgG or anti-NRF2 antibody (Genetex, San Antonio, TX, USA). The levels of targeted genes in ChIP products were determined by RT-qPCR. Primers used are listed in Table S1.

One hour after UVB exposure, Nrf2 intracellular localization was assessed by immunofluorescence assays. For this experiment, 7 × 10³ cells were seeded on 12-mm coverslips (Menzel-Gläser, Waltham, MA, USA). After a 30-min fixation at RT with 4% paraformaldehyde (PFA, PanReac AppliChem, Barcelona, Spain), cells were permeabilized and non-specific protein interactions were blocked by incubation with 1× PBS, containing 3% BSA and 0.1% Triton X-100, for another 30 min at RT. Cells were then incubated overnight at 4 °C with the primary antibody (anti-Nrf2 antibody, 1:500, Genetex, Irvine, CA, USA). After incubation, cells were washed three times with 1× PBS and incubated with a secondary antibody (Goat anti-Rabbit IgG Alexa 488, 1:2000, Invitrogen, Eugene, OR, USA) for 1 h at RT in the dark. Finally, after three more times washes, cells were observed under a Zeiss LSM800 confocal microscope (Carl Zeiss, Chicago, IL, USA) using a ×20 objective.