Protein Extraction and Quantification from Liver Tissue

Protein was extracted from liver tissue or cell cultures as described.^{2 (link)} Protein was extracted from liver tissue or cell cultures with ice-cold protein lysis buffer (50 mM Tris, 150 mM NaCl, 0.1% sodium dodecyl sulfate, 1% sodium deoxycholate, and 1% Triton-100). Proteins (30 µg/sample) in SDS-loading buffer (50 mM Tris, pH 7.6, 10% glycerol, and 1% SDS) were subjected to 4–20% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA). The membrane was blocked with 5% dry milk and 0.1% Tween 20 (USB, Cleveland, OH). Monoclonal rabbit anti-mouse NICD, HSF1, Snail, TRX1, TXNIP, NLRP3, ASC, cleaved caspase-1, p-ASK1, ASK1, Bcl-2, Bcl-xL, cleaved caspase-3, and β-actin Abs (Cell Signaling Technology, MA) were used. The membranes were incubated with the Abs and then developed according to the Pierce SuperSignal West Pico Chemiluminescent Substrate protocol (Pierce Biotechnology, Rockford, IL). The relative quantities of protein were determined using a densitometer (Kodak Digital Science 1D Analysis Software, Rochester, NY) and expressed in absorbance units (AU) by comparison with β-actin expression.