Fig. 6.
AP-1 binding and c-Jun protein levels in affected DRGs after distal ligation with cuff or sciatic nerve crush. At the indicated times after mid-thigh sciatic nerve transection with cuff (a, b) or crush (c, d), injured (I) or contralateral uninjured (C) DRGs were analyzed by EMSA with Hmt IIA AP-1 oligonucleotides (a, c) or enhanced chemiluminescent Western blotting for c-Jun protein (b, d). Shown are autoradiographs representative of samples analyzed from three rats at each time point, for each surgery. All animals were included in quantitative densitometric analysis described in Results. The level of injury-induced AP-1 binding activity in each sample was determined as a ratio of AP-1 binding activity in injured versus contralateral, uninjured samples. The participation of c-Jun in AP-1 binding activity was determined by the ability of an antibody against the c-Jun DNA binding domain to inhibit formation of AP-1 binding complexes. Samples treated with the c-Jun antibody are indicated above the lane (*). The specific AP-1 binding complex signal is shown by <. Samples analyzed by EMSA were also immunoblotted for c-Jun, under previously optimized experimental conditions that have been established to quantify changes in c-jun protein levels in axotomized DRGs using chemiluminescent detection (Kenney and Kocsis, 1997, 1998), also described in Materials and Methods. The c-Jun antibody used recognizes four major species: 80, 60, 46, and 30 kDa. Only the doublet appearing at 46 kDa is regulated by axonal transection. When extracts from UV-irradiated NIH 3T3 cells were immunoblotted for c-Jun using this antibody, the same hybridization pattern was observed, and the 46 kDa doublet was the only species induced by UV light. Therefore, this species was understood to represent c-Jun and was quantified by densitometric analysis of ECL autoradiographs. A ratio of c-jun immunoreactivity in injured versus the contralateral uninjured DRGs was then established to determine axotomy-induced changes in c-jun protein levels.