Figure 3

The effect of 8R-sJNKI(-9) on porcine islets. (a) Western blot. Cell extracts were fractionated by 10% SDS-PAGE and transferred to reinforced cellulose nitrate membrane. After blocking, the membranes were incubated overnight at 4 °C in TBS buffer containing a 1:1,000 dilution of rabbit anti-phospho-c-Jun antibody, mouse anti-JNK1 antibody, or mouse anti-β-actin antibody (control), and then incubated for 1 h at room temperature in TBS containing a secondary antibody. unt: untreated islets, sJNKI: islets treated with 8R-sJNKI(-9), mJNKI: islets treated with 8R-mJNKI(-9). (b,c) Propidium iodide (PI)/annexin-V assays. After 6-h culture with or without 1 µM of 8R-sJNKI(-9) or 1 µM of 8R-mJNKI(-9), 1,000 IE of islets were dispersed into single cells using Accutase and the single cells were then incubated with PI (b) and FITC-annexin-V (c). (d,e) Propidium iodide (PI)/annexin-V assays after exposure to cytokines. Islets that had been cultured for 48 h were treated with or without 8R-sJNKI(-9) or 8R-mJNKI(-9) for 23 h and then exposed to a cocktail of cytokines (IL-1β 50 U/mL, TNF-α 1,000 U/mL, IFN-γ 1,000 U/mL) for 18 h., 1,000 IE of islets were dispersed into single cells using Accutase, and the single cells were then incubated with PI (d) and FITC-annexin-V (e). The data are representative of six independent experiments.