Quantification of Endoplasmic Reticulum Stress Markers by Western Blotting

Isolated islets were cultured for 1 day, and as a positive control, one group of wild-type islets were then treated for 16 h with 1 μm thapsigargin to induce endoplasmic reticulum (ER) stress (10). Islets pooled from two to three mice in each condition were lysed using radioimmunoprecipitation assay buffer supplemented with a protease and phosphatase inhibitor cocktail. Fifteen micrograms of protein per sample were subjected to SDS-PAGE and transferred to a polyvinylidene fluoride membrane. Membranes were treated with antibodies toward BiP, XBP1s, p-eIF-2-α (Cell Signaling Technology) and β-actin (Santa Cruz Biotechnology). Densitometry analysis was performed on Western blot images using ImageJ software.