2.4. Immunoblotting Analysis

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).