Molecular weight standards

Cells were homogenized with cold RIPA lysis buffer followed by 3 cycles of 20-s bursts of sonication at 4°C. The amount of protein per sample was determined using a dye-binding protein assay (Bio-Rad). Electrophoresis was performed on the samples using 4–12% or 10% acrylamide gels (Invitrogen) and transferred to polyvinylidene difluoride membranes (Millipore) by electroelution. Membranes were blocked in blocking solution [20 mM TBS Tween (1%) (TBST) containing 3% bovine serum albumin (BSA)] and then incubated with primary antibodies overnight at 4°C. After 3 washes with TBST, the membrane was incubated with secondary antibody for 1 h at room temperature (RT). Horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (Santa Cruz Biotech) was used as secondary antibody. After another 3 times wash with TBST to remove the rest secondary antibody, membrane was incubated with ECL reagent (Amersham Pharmacia Biotech, Piscataway, NJ, United States) and prepared for imaging. Bands were compared to molecular weight standards (Santa Cruz Biotech). GAPDH served as the internal reference by which to standardize the other protein bands. The calculated ratio of the control group was normalized to 100%, and the comparisons of other groups to the control group were represented as percentages.

Proteins were separated by SDS-PAGE 10% polyacrylamide precast gels (Bio-Rad Laboratories) and transferred to a polyvinylidene diflouride membrane by electroelution. Membranes were blocked in PBS containing 2.0% nonfat dry milk and incubated with primary antibody overnight (GLP-1R and caveolins-3, Santa Cruz Biotechnology; GAPDH, Santa Cruz Biotechnology and Cell Signaling Technology) and at 4°C. Bound primary antibodies were visualized using secondary antibodies (Santa Cruz Biotechnology) conjugated with horseradish peroxidase from Santa Cruz Biotechnology and ECL reagent from GE Healthcare [24 (link)]. All displayed bands migrated at the appropriate size, as determined by comparison to molecular weight standards (Santa Cruz Biotechnology).