Fig. 6. Gene transfer of IB1/JIP-1 can prevent the JNK activity. (A) Western blot analysis of scraped urothelial cells infected with different adenovirus constructs showed that IB1/JIP-1 content is largely increased in cells overexpressing IB1/JIP-1 (adCMV-sIB1), whereas a decreased expression was observed when cells were infected with the adCMV-asIB1 compared to control adCMV-GFP. Protein loading was evaluated by tubulin. (B) JNK activity analysis in urothelial cells of dilated bladder revealed that cells infected with adCMV-asIB1 increased their JNK activity; in contrast, cells infected with adCMV-sIB1 decreased their JNK activity. Quantitative assessment demonstrated that the JNK activity is increased about three-fold in urothelial cells of dilated bladder infected with adCMV-asIB1 compared with non infected bladder (NI) or infected with control adenovirus (adCMV-GFP). In contrast, a 50% reduction in JNK activity is observed in cells infected with adCMV-sIB1. In the same samples, an increase in P-JNK was detected by immunoblot in urothelial cells infected with adCMV-asIB1; in contrast, a decrease in P-JNK content was observed in cells infected with adCMV-sIB1 compared to controls (NI, adCMV-GFP). (C) Electrophoretic mobility shift assay (EMSA) revealed a decrease in AP-1 binding activity in stress urothelium transduced with adCMV-sIB1/JIP-1 compared to non-infected dilated urothelium (NI) or cells transduced with the control adenovirus. (D) Western blot analysis of c-Jun expression on the same nuclear extract of dilated urothelial cells demonstrated a decrease in c-Jun expression in bladder transduced with adCMV-sIB1 compared to non infected bladder (NI) or infected with the control adenovirus.
