Halt protease inhibitor cocktail

hCMs were washed with ice-cold PBS and lysed in TX-100 RIPA buffer with protease inhibitor (Halt™ Protease Inhibitor Cocktail (100×)), phosphatase inhibitor (Halt™ Phosphatase Inhibitor Cocktail (100×)) and benzonase nuclease (Santa Cruz Biotechnology). Protein concentrations were determined with Pierce BCA Protein Assay Kit (Thermo Scientific) and lysates were treated with 5× sample buffer (312.5 mM Tris, pH  = 6.8, 50% glycerol, 0.37 mM bromphenol blue, 347 mM SDS, 2.5% β-mercaptoethanol). Equal protein amounts were separated by SDS-polyacrylamide gel electrophoresis and transferred to 0.2-µM nitrocellulose membranes (GE Healthcare). Membranes were blocked in 5% BSA for 1 h. Primary antibodies (NRF2 sc-722, Santa Cruz, 1:500; GAPDH 14C10, Cell Signaling, 1:10,000) were diluted in blocking solution and incubated overnight (4 °C). HRP-conjugated secondary anti-rabbit antibodies (P0448, Dako, 1:5000) were incubated for 1 h at RT. ECL detection (Merck Millipore) was used for visualization with the Amersham Imager 600 (GE Healthcare). Band intensities were quantified in ImageQuant TL (GE Healthcare). The uncropped blots are depicted in the source data file.

Cell was scraped and centrifuged at 4 °C. Pellets were lysed in RIPA Lysis Buffer System supplemented with 1 × Halt protease inhibitor cocktail and 1 × Halt phosphatase inhibitor cocktail (Santa Cruz Biotechnology). Protein concentrations were measured using Bio‐Rad protein Assay (Bio‐Rad). Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS/PAGE) and transferred to Protran nitrocellulose membranes (Whatman, Boston, MA, USA). Membranes were probed with specific primary antibodies, followed by horseradish peroxidase (HRP)‐conjugated secondary antibodies (Promega). Protein bands were visualized using Amersham ECL western blotting detection reagent (GE Healthcare, Pittsburgh, PA, USA), and images were taken on the ChemiDoc XRS System (Bio‐Rad). The protein bands intensities were semiquantitatively analyzed by densitometry using imagej software (NIH Image).