Substrate detection kit

Whole cell protein lysates were prepared using RIPA buffer containing 0.2% SDS, 1 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate, 1 mM sodium orthovanadate, 1 mM sodium fluoride, 1 mM PMSF and EDTA-free mini-complete protease inhibitor cocktail tablet (Roche). Tissue samples or cells were homogenized in RIPA Lysis Buffer. The protein concentration was determined using Bradford’s Reagent (BioRad). 40 µg of protein was subjected to polyacrylamide gel electrophoresis and transferred on to a nitrocellulose membrane (Merck Millipore). Membranes were incubated with respective primary antibody dilutions prepared in Tris Buffered Saline (TBS)-Tween for 2 h at room temperature or overnight at 4 °C. Membranes were then washed in TBS-Tween and incubated with secondary antibodies anti-rabbit IgG–HRP (BioRad) or anti mouse IgG-HRP (BioRad) diluted (1:10.000) in TBS-Tween for 1 h at room temperature. Protein-antibody complexes were detected by Substrate Detection Kit (Thermofischer, USA). Quantification of signal was done via densitometry analysis, using the Image J software. Actin was used as loading control for whole cell lysates and Lamin A/C and GAPDH were used as loading controls for nuclear and cytoplasmic fractions respectively.

Protein extracts were prepared, and immunoblotting was performed, as described previously [15 (link)], using the following antibodies: Nrf2, Bach1, SNAIL and SLUG (Abcam, Burlingame, CA, USA); NQO1, HO1 (Santa Cruz, TX, USA), Keap1, SOX2 and KLF4, NANOG (Cell Signaling Technology, ((Danvers, MA, USA); BAX and BCL2 (Santa Cruz, Dallas, TX, USA) at 1:1000 and GAPDH (1:10,000, Sigma). Primary antibodies were detected using secondary antibodies (1:10,000, Bio-Rad), conjugated with HRP, and protein–antibody complexes were detected by the Substrate Detection Kit (Thermo Fisher, CA, USA). Densitometry was performed using the Image Lab and Image J software. GAPDH was used as loading control for whole-cell lysates.

Western blotting was performed as described previously [30 (link)], using the antibodies described in Table S3. The Substrate Detection Kit detected protein–antibody complexes (Thermo Fisher, Carlsbad, CA, USA). GAPDH or β-tubulin was used as a loading control.