Nrf2 antibody

TR146 cells were treated as described above. Cells were fixed with 4% paraformaldehyde followed by permeabilisation with 0.02% Triton-X for 20 min. After washing with PBS, the cells were blocked with 5% bovine serum albumin (Sigma-Aldrich, Missouri, USA) for 1 h at room temperature, followed by incubation with Nrf-2 antibody (Abcam) overnight at 4 °C. Subsequently, the wells were washed and incubated with goat anti-rabbit IgG-Alexa Flour 488 (Abcam) for 1 h and counterstained with 4′6-diamidino-2-phenylindole, DAPI (Abcam) and Phalloidin-iFlour 594 reagent (Abcam) for 5 min each. Stained wells were analysed using a Leica DMi8 fluorescent microscope.

Kidney cortex and heart were homogenized in cell lysis buffer (Cell Signaling, Danvers, MA) and Western blotting was performed as described previously^{19 (link)}. Briefly, samples were subjected to SDS-PAGE and transferred onto a nitrocellulose membrane. After overnight incubation with primary antibody at 4 °C, membrane was incubated with secondary antibody linked with horseradish peroxidase for 1 h at RT. Signal was detected by Immun Star HRP (Bio-Rad, Hercules, CA). The density of each band was determined using Multi Gauge software (Fuji film, Tokyo, Japan) and expressed as a value relative to the density of the corresponding band of the GAPDH. As primary antibodies, Nrf2 antibody (abcam, Cambridge, UK) and heme oxygenase-1 (HO-1) antibody (Enzo Life Sciences, NY, USA) were used. Full-length blots are shown in Supplemental Fig. 1.

After JB6 P+ cells seeded and grown on glass cover slips were treated by indicated agents, they were fixed by pre-cold acetone, then rinsed three times with 1× phosphate-buffered saline (PBS). The cells were permeabilized in 0.1% Triton X-100 and incubated with 1% bovine serum albumin (BSA) in 1× PBS to block nonspecific binding. Subsequently, the cells were immunostained by incubating with Nrf2 antibody (1: 100, Abcam, Cat.NO. ab137550) overnight at 4°C. After being washed with PBS, cells were incubated with Rhodamin-conjugated goat anti-rabbit antibody (1: 200, CWBIO, China, Cat.NO. BA1105). Actin filaments were stained using Actin-Tracker Green (Beyotime, China). Nuclei were counterstained with Hoechst 33258 (Beyotime, China). Fluorescent images were taken and analyzed using the ZEN pro 2012 imaging software on a Zeiss invert microscope under 200-fold magnification.

N2a/APPswe cells treated with or without 5-Aza were fixed with 4% paraformaldehyde for 15 min at room temperature on the slides and followed by a rinse with phosphate-buffered saline (PBS) three times for 15 min. After washing, cells were incubated with 0.3% Triton for 15 min at room temperature and then blocked with 5% BSA for 30 min. The cells were incubated with Nrf2 antibody at 1:100 (Abcam, USA) overnight at 4°C. Next day the cells were washed three times in PBS for 15 min. Then, the cells were incubated with goat anti-rabbit conjugated to DyLight 549 secondary antibody at 1:200 (Abbkine, USA) for 30 min at 37°C. Later, the cells were washed three times in PBS for another 15 min and then stained with DAPI (Beyotime, Dalian, China) for 3 min at room temperature. Finally, cells were washed in PBS for 15 min and observed with a Confocal Laser-scanning Microscope (A1R, Nikon, Japan).

After JB6 P+ cells on glass coverslips were treated by indicated agents, they were fixed by pre-cold acetone, then rinsed three times with PBS. The cells were permeabilized in 0.1% Triton X-100 and incubated with 1% BSA/PBS to block nonspecific binding. Subsequently, the cells were immunostained by incubating with Nrf2 antibody (1:100, Abcam) overnight at 4 °C. After being washed with PBS, cells were incubated with Rhodamin-conjugated goat anti-rabbit antibody (1:200, CWBIO, China). Actin filaments were stained using Actin-Tracker (Beyotime, China). And nuclei were counterstained with Hoechst 33258 (Beyotime, China). Fluorescent images were taken and analyzed using the ZEN pro 2012 imaging software on a Zeiss invert microscope under 400-fold magnification.

Validation of different expression proteins (NQO1, ALDH1A1, GSR, CDK4 and G6PD, GCLC, GCLM, GSTP1, NRF2) in different cell lines was processed on a traditional way. Briefly, separated denatured proteins on a SDS-PAGE and transferred onto a PVDF membrane, Incubated with primary antibody (1000 × diluted) and secondary antibody (2000 × diluted) sequentially after blocking. The membrane was further washed with PBST and developed using ECL reagents (Pierce, USA). GAPDH was detected as control. The primary antibody of NQO1, ALDH1A1, GSR, CDK4 and G6PD, GCLC, GCLM, GSTP1was purchased for Ptglab, and the NRF2 antibody was purchased from Abcam.

Chromatin was immunoprecipitated with p53 DO-1 (Santa Cruz) or NRF2 antibody (Abcam), as previously described [14 (link)]. As negative controls IgGs purified from rabbit or mouse serum were used. Coimmunoprecipitated DNA was analyzed by real-time PCR on a CFX96 Real-Time PCR System (Biorad), using SsoAdvanced™SYBR Green Master Mix (Biorad). Promoter occupancy was calculated as percent of input chromatin immunoprecipitated using the 2^-DCt method. Primer sequences are shown in the Supplementary Table 3.