Nupage western blotting system

Islets were isolated from 8-week-old animals as described above. Following isolation, islets were immediately lysed in RIPA buffer, and protein content was quantified using the Bio-Rad DC protein assay (Bio-Rad). A total of 3.5 μg of protein per sample was electrophoresed on 4–12% Bis-Tris gels under denaturing conditions and blotted onto polyvinylidene difluoride membrane using the NuPAGE Western blotting system (Invitrogen). Blots were blocked and probed with the following primary antibodies diluted in 5% nonfat milk in 1x Tris-buffered saline with Tween and incubated overnight at 4°C: rabbit anti–phospho-Smad3 (1:1,000; Abcam), rabbit anti-Smad2/3 (1:500; Cell Signaling Technology), and rabbit anti–β-tubulin (1:5,000; Santa Cruz Biotechnology). Horseradish peroxidase–conjugated rabbit secondary antibody (1:5,000; Jackson ImmunoResearch Laboratories) was used for protein detection and facilitated by an ECL Prime detection system (Amersham) using Kodak X-Omat Blue film. Protein levels were quantified using ImageJ software.