Fig. 4. Phosphorylated JNK activity is functionally active and associated with an increase in AP-1 binding activity. (A) JNK activity was evaluated in urothelial cells. Endogenous JNK was first purified using GST-Jun, and then a solid phase kinase assay was performed. [-³³P]-phosphorylated substrates (P-GST-Jun) were separated on a polyacrylamide gel. Western blotting monitored JNK expression in the same samples. The gel was stained with Coomassie blue to evaluate the loading of substrate (GST-Jun). P-GST-Jun showed a progressive increase during the development of the dilated bladder. Maximum expression occurred seven hours after ligature. All lanes were loaded with 50 µg of protein. (B) JNK activity in urothelium was monitored seven hours after ligature. Quantitative assessment demonstrated that urothelial cells of dilated bladder increased c-Jun phosphorylation about five fold compared to the empty bladder. On the same samples, IB1/JIP-1 content was decreased in freshly scraped urothelial cells from the dilated bladder. Values represent densitometric measurements of six independent experiments and are expressed as mean±s.e.m. ***=P<0.001 level. (C) Electrophoretic mobility shift assay (EMSA) showed that AP-1 binding activity is increased in stressed urothelium seven hours after ligature. Each lane shows a sample from a different rat. All lanes were loaded with 7 µg. (D) Specificity of the AP-1 shift. Nuclear extracts of freshly scraped urothelial cells were pre-incubated with specific c-Jun antibody (c-Jun) or two different irrelevant antibodies (IgG). AP-1 shift was competed with the incubation of a cold-specific competitor (specific) but not with non-specific oligonucleotides (NS). (E) Western blot analysis of c-Jun expression on the same nuclear extract of freshly scraped urothelial cells used for EMSA experiment seven hours after ligature demonstrated a 400% increase of c-Jun expression in dilated bladder compared with the control.