Protein Expression Analysis in Hepatocytes

The treated human LO-2 hepatocytes were lysed to isolate proteins, which were subsequently loaded and separated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Then, the proteins were transferred onto the polyvinylidene difluoride (PVDF) membrane and incubated with 5% nonfat milk. The membrane was then incubated with primary antibody against NOX-4 (1:1000, ab133303, Abcam), NLRP3 (1:1000, ab263899, Abcam), p-eIF-2α (1:1000, ab169528, Abcam), ATF4 (1:1000, ab184909, Abcam), GADD34 (1:1000, ab236516, Abcam), CHOP (1:1000, ab11419, Abcam), NF-κB p65 (1:1000, ab288751, Abcam) or β-actin (1:1000, ab8226, Abcam), which was further incubated with an HRP-conjugated secondary antibody. Finally, the blots were developed with electrochemiluminescence (ECL) reagents and analyzed using Image J software [25 (link)].