Sc 13032x

A549 cells were harvested using a cell scraper and washed three times in 1x PBS. The cell pellet was resuspended in 1x PBS containing 0.5 mM DTME and 0.5 mM DSP and incubated at room temperature for 30 min, followed by incubation in quenching buffer (20 mM Tris-HCl (pH 7.5), 5 mM cysteine) at 25 °C for 5 min. After washing in ice-cold PBS, the pellet was sonicated in RIPA buffer briefly and centrifuged at 14,000 × g at 4 °C for 5 min. The supernatant was subjected to anti-NRF2 affinity purification. An anti-NRF2 antibody (#12721, Cell Signaling Technology) and control rabbit IgG (#55944, Thermo Fisher Scientific) were crosslinked to a 1:1 mixture of Dynabeads protein A and protein G (Thermo Fisher Scientific) with DMP and incubated with the supernatant at 4 °C for 2 h. After washing in RIPA buffer, the NRF2 complex was eluted from the beads by incubation in elution buffer (50 mM Tris-HCl (pH 8.0), 0.2 M NaCl, 2 w/v% SDS, 50 mM DTT) at 37 °C for 30 min. The eluate was analyzed by immunoblot analysis with anti-NRF2 antibody (sc-13032X, Santa Cruz; 1:1,000), anti-FOSL2 antibody (#19967S, Cell Signaling Technology; 1:1,000), and anti-CEBPB antibody (sc-150 X, Santa Cruz; 1:1,000).

For the preparation of nuclear lysates, cells were lysed in buffer A (10 mM HCl (pH7.5) 10 mM KCl, 1.5 mM MgCl₂, 0.1% NP40), and crude nuclei were pelleted by centrifugation and lysed in 2x Laemmli buffer followed by boiling at 95 °C for 5 min. For the preparation of whole-cell lysates, cells were directly lysed in 2x Laemmli buffer followed by boiling at 100 °C for 10 min. The protein samples were separated by SDS-PAGE and transferred onto PVDF membranes (Immobilon P, Millipore). The antibodies used are as follows: anti-NRF2 (sc-13032X, Santa Cruz; 1:1,000-1:500), anti-KEAP1 (#111; 1:200)^{58 (link)}, anti-NOTCH3 (ab23426, Abcam; 1:2,000), anti-CEBPB antibody (sc-150 X, Santa Cruz; 1:1,000), anti-Tubulin (T9026, Sigma; 1:5,000-1:2,000) and anti-Lamin B (sc-6217, Santa Cruz; 1:2,000). Quantification of band intensities in the immunoblot data of human samples were conducted by ImageJ software (ver. 1.45 s; http://imagej.nih.gov/ij). Pearson’s correlation coefficient was calculated for correlation of band intensities between NRF2 and CEBPB.