Fig. 6.

Mutation of the GC-box decreases Sp3 binding yet can enhance cAMP-dependent transcription in PC12 cells. (a) EMSA using 1 μg of PC12 nuclear protein and 1 μg of control IgG, anti-Sp1 or anti-Sp3 shows that mutation M1 of the GC-box does not affect formation of the Sp1- and Sp3-containing complexes 3, 4 and 5 or complex 6 but does eliminate complexes 1 and 2, indicating that these complexes contain Sp3. Mutation M1234 completely eliminates PC12 nuclear protein binding to the GC-box. (b) Luciferase assays of PC12 cells transiently transfected with the wild-type (WT) 400 ng of p0.27GCH1-GL3 or reporter constructs containing the GC-box mutations M1, M2, M3, M4, or M1234, 40 ng of pRL-null and 360 ng of carrier plasmid DNA and then challenged with 5 mmol/L 8Br-cAMP (cAMP) for 4 h. The insert shows control activity plotted on a smaller scale. Data were analyzed by ANOVA with post hoc Bonferroni tests (*p < 0.05 vs. WT control or cAMP).