Figure 3.

A p65 mutant lacking spine enrichment is selectively deficient in response to synaptic stimulation. A, Inherent transactivation potential of wild-type p65 and p65 lacking spine enrichment (p65ΔSE) are not distinguishable. Reporter assay for dose-titrated expression of GFPp65 or GFPp65ΔSE in HEK293T cells coexpressing NF-κB luciferase reporter and constitutive β-galactosidase used for normalization (left). Fold induction calculated as described in Materials and Methods. Representative immunoblot of dose titration (right). B, NF-κB luciferase assay of p65-deficient neurons expressing full-length GFPp65 or GFPp65ΔSE and either mock-stimulated or stimulated with bicuculline (30 μm for 30 s), with ionomycin/PMA (3.5 h), or with glycine (100 μm for 10 min). *p = 0.018 compared with GFPp65 no stimulation. **p = 0.0002 compared with GFPp65ΔSE no stimulation. ****p = 0.0001 compared with GFPp65 no stimulation. For neurons expressing GFPp65ΔSE, NF-κB reporter activity in response to bicuculline or glycine does not differ significantly from the no stimulation condition. Representative immunoblot (inset) illustrating similar levels of expressed protein for GFPp65 or GFPp65ΔSE in these experiments. C, NF-κB luciferase assay of wild-type neurons either mock-stimulated or stimulated with ionomycin/PMA (2 μm and 50 ng/ml), bicuculline (30 μm for 30 s), or with glycine (150 μm for 10 min) and treated with either TPCA1 (4 μm) or TAT-NBD (20 μm) or their respective vehicle or TAT controls. *p = 0.04 compared with no stimulation, which was set to 1. ***p = 0.0007 compared with no stimulation, which was set to 1. ****p = 0.0001 compared with no stimulation, which was set to 1. Error bars indicate SEM.