Image quant apparatus

Groups of 350 islets were resuspended in 80 µL extraction buffer (100 mM Tris, 1% Triton X-100, 0.01 mg/mL aprotinin, 2 mM PMSF, 10 mM Na 3 VO 4 , 10 mM NaF, 10 mM Na 4 P 2 O 7 and 10 mM EDTA). The samples were boiled for 5 min and centrifuged for 20 min at 4°C (10,000 g). The protein content of the supernatant was determined by the Bradford method (1976). For immunoprecipitation (IP), pancreatic islet extracts were centrifuged for 20 min at 4°C (12,000 g). The supernatant (3 mg of protein) was used for IP with anti-p47phox and protein A-Sepharose 6 MB before Laemmli sample buffer containing 100 mM dithiothreitol. After running gel electrophoresis (10% gel) and transferring to PVDF membrane, expression of the subunits of NADPH oxidase 231:3 was assessed using the antibodies described in Table 1. The enhanced chemiluminescence reagents -ECL -(GE Healthcare) were used, and the membrane was exposed to photographic film or Image Quant apparatus, GE Healthcare (GE Healthcare AS). The intensity of the bands was quantified by optical densitometry using ImageJ software (Wayne Rasbond, NIH, Bethesda, MD, USA). Quantification of protein expression was normalized by protein α-tubulin.