Alpha imager 3300 system

To measure protein expression of Kir6.1, frozen tissue from the AAR was thawed, finely minced and homogenized. The tissue homogenate was boiled in NuPAGE LDS sample buffer (Invitrogen) for 10 min. Protein homogenates were subjected to electrophoresis on NuPAGE Novex 4–12% Bis-Tris gels (Invitrogen) and transferred onto nitrocellulose membranes (Amersham Biosciences). After blocking with 5% non-fat milk in Tween-20- and Tris-buffered saline (TTBS) for 1 h at room temperature, the membranes were incubated with goat polyclonal antibodies against Kir6.1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1∶1000, Santa Cruz Biotechnology). After incubation, the membranes were washed with TTBS and exposed to the antibodies conjugated with horseradish peroxidase for 1 h at room temperature. Proteins were detected using chemiluminescence Western immunoblotting detection reagents (Amersham Biosciences). Densitometric analyses of the immunoblots were performed using an Alpha Imager 3300 system (Alpha Innotech, San Leandro, CA).