Anti pire1α

Tissues were homogenized using TissueLyzer II (Qiagen, Hilden, Germany). Cells and tissues were lysed using the radioimmunoprecipitation assay (RIPA) buffer (30 mM Tris [pH 7.5], 150 mM sodium chloride, 1 mM sodium phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 1% Nonidet P-40, 10% glycerol, and phosphatase and protease inhibitors). Western blot analyses were performed with 40 μg of protein, using commercially available antibodies. Anti-CHOP, anti-pERK, anti-ERK, anti-IRE1α, anti-pIRE1α, anti-pSMAD2/3, and anti-β-actin antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA). Anti-NDUFA, anti-SDHA, anti-UQCR2, and anti-ATP5A antibodies were purchased from Abcam (Cambridge, UK). Anti-COL1A2, anti-COL1A1, anti-ATF4, anti-TGF-β1, and anti-IL-1 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Peroxidase-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology. Immunoreactive bands were visualized using enhanced chemiluminescence (ECL; Bio-Rad, Hercules, CA, USA). Images were scanned using the Odyssey imaging system and quantified using Image Studio Digits (LI-COR Biosciences, Lincoln, NE, USA).

The samples were then homogenized with ice-cold ProEX™ CETi protein extract solution (TransLab Biosciences, Daejeon, Korea), which contained a protease and phosphatase inhibitor cocktail. The concentration of protein in the lysates was determined using the BCA procedure (Thermo Scientific, Sunnyvale, CA, USA). The same amounts of protein were separated and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The samples were then transferred onto nitrocellulose (NC) membranes (Bio-Rad, Hercules, CA, USA). The membranes were incubated with 0.1% Tween-20 (TBS-T), which contained 5% skimmed milk for 30 min at 25 °C. They were then washed with TBS-T buffer. The following primary antibodies were incubated overnight at 4 °C (dilution 1:2000 to 1:5000): anti-IRE1α, anti-p-IRE1α (Cell Signaling Technology, Danvers, MA, USA), anti-ATF6, anti-CHOP, anti-GCLC, anti-GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-CD36 (Abcam, Cambridge, MA, USA), and anti-CDO (Abcam, Cambridge, MA, USA). The blots were washed with TBS-T and incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies. The resulting antigen-antibody complexes were detected using the EZ-Western Lumi Pico detection kit (DOGEN, Seoul, Korea).