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. 1998 Mar 1;18(5):1713–1724. doi: 10.1523/JNEUROSCI.18-05-01713.1998

Fig. 1.

Fig. 1.

Transcriptional activation of the c-jun promoter in NGF-deprived sympathetic neurons requires the jun1 and jun2 TRE elements and AP-1 activity.A, Sympathetic neurons were injected with pCDFLAGΔ169 (0.2 mg/ml) or the empty CMV expression vector pcDNA1 (0.2 mg/ml) together with guinea pig IgG (2.5 mg/ml). After injection, the cells were rinsed with medium lacking NGF and then were refed with −NGF medium supplemented with neutralizing anti-NGF antibody. Twenty-four hours later, the percentage of injected cells that expressed c-Jun protein was determined as described in Materials and Methods. Cells were only considered to be expressing c-Jun if nuclear staining with the c-Jun antibody was more intense than the background cytoplasmic staining. In each experiment 200 cells were injected per construct. Uninjected cells on the same coverslips were also scored for comparison. Coverslips were scored blind. The data shown represent the average of three independent experiments. Error bars indicate SE.B, Reporter gene structure. c-jun CAT contains wild-type c-jun promoter sequences from −1600 to +170 cloned upstream of the bacterial CAT gene. The position of the jun1 and jun2 TRE elements is indicated (j1j2). In j1j2 CAT, these have been mutated so that they are nonfunctional. 6xjun2 SVeCAT was constructed by cloning six copies of the jun2 TRE element upstream of the SV40 early promoter in SVeCAT (= pCAT3 promoter). C, c-jun CAT or j1j2 CAT was microinjected into sympathetic neurons at a concentration of 0.01 mg/ml together with guinea pig IgG (2.5 mg/ml). The injected cells were refed with medium containing (+NGF) or lacking (−NGF) NGF. Eighteen hours later, the percentage of cells expressing CAT was determined in immunofluorescence experiments with an anti-CAT antibody as described in Materials and Methods. The data shown represent the average of three independent experiments. Error bars indicate SE. D, 6xjun2 SVeCAT or SVeCAT was microinjected into sympathetic neurons at a concentration of 0.005 mg/ml together with guinea pig IgG. The injected cells were treated as described in C.