Protease inhibitors cocktail

Western blot assay was conducted as previously described [17 (link)]. The proteins from the cell were lysed in RIPA buffer (pH = 7.4) comprised of protease inhibitors cocktail (10 μg/ml, Cat# 5872S, Cell Signaling Technology, MA, U.S.A.). The contents were centrifuged at 12,000 g at 4°C for 20 min, and the concentration of protein in the supernatants was determined using Bradford reagent (BioRad) with bovine serum albumin (BSA, Sigma Aldrich, St. Louis, MO, U.S.A.) as the standard. The protein samples were separated by electrophoresis on SDS-PAGE 10% or 12.5% gels. After blocked in 3% BSA, the membrane was incubated with primary antibodies, GAPDH (Cat# 2118 s, Cell Signaling Technology), caspase-1 (Cat# sc-56036, Santa Cruz Biotechnology, U.S.A.), GSDMD (Cat# 93709 s, Cell Signaling Technology), JNK1/2/3 (Cat# YT2440, Immunoway, TX, U.S.A.), caspase-3 (Cat# sc-7148, Santa Cruz Biotechnology), caspase-9 (Cat# 9502, CST), caspase-4 (Cat# ab238124, Abcam, Cambridge, United Kingdom), cleaved caspase-3 (Cat# 9661 s, Cell Signaling Technology), and GSDME (Cat# ab215191, Abcam) as needed. For secondary antibodies, antibodies to mouse (Cat# 7076, Cell Signaling Technology) and rabbit (Cat# 7074, Cell Signaling Technology) were used. To visualize protein bands, a chemiluminescence (ECL) system (Cat# WBLUF0500, Millipore, MA, U.S.A.) was used.

Catechol-modification of proteins was assessed in DOPAL-treated cells as described in Mazzulli et al. with slight modification^{29 (link)}. Untreated BE(2)-M17 cells and DOPAL-treated cells were harvested in radioimmunoprecipitation assay (RIPA) buffer (Cell Signaling Technology) supplemented with protease inhibitors cocktail (Roche). After lysates centrifugation at 20,000 × g at 4 °C, the cleared supernatant was collected and the protein content in the soluble fraction was determined using the Pierce® BCA Protein Assay Kit (Thermo Scientific) following the manufacturer’s instructions. The insoluble fraction in the pellet of the cell lysate was resuspended in 1 M NaOH (volumes were adjusted according to the protein quantification in the soluble fraction), followed by incubation at 37 °C for 30 min, sonication at room temperature for 20 min (bath sonicator, 100% power and 80 Hz frequency) and centrifugation at maximum speed at room temperature for 20 min. The pellets were then dried at 95 °C and resuspended in 10 µl of water. Both the soluble (40 µg of proteins) and the insoluble (4 µl) fractions were spotted on nitrocellulose membrane (BioRad). The near infrared fluorescence (nIRF) signal was acquired by scanning the membrane at the LiCor Odissey using the 800 nm filter and images were analyzed using the Fiji software.

Homogenized lung tissues were lysed with lyses buffer containing protease inhibitors cocktail (Cell Signaling, Boston, MA, USA). BCA method was used to determine protein concentration (Pierce Chemical Co., TX, and USA). Total 50 μg protein samples were separated by 10% sodium codicil sulfate-PAGE, and were transferred to a hyoid-enhanced chemiluminescence’s (ECL) nitrocellulose membrane. The membrane was blocked in 5% skim milk powder at room temperature for one hour. Then, the membrane was blotted with the primary antibody (1:1000) against Nrf2 (Cambridge, MA, USA) and Kelch-like ECH-associated protein 1 (Keap1) (Santa Cruz Biotechnology Inc., Santa Cruz, CA,USA) overnight at 4 °C and the corresponding secondary antibody (1:5000) at room temperature for one hour. Finally the blots were visualized with an enhanced ECL western blot detection system (Habersham Pharmacia Biotech, Piscataway, NJ, USA). The band intensities were quantified using Image J 1.47v software (NIH.USA). Laming B and β-acting (Cell Signaling, Boston, MA, USA) were used as internal references for determination of nuclear and cryptozoic protein, respectively.