Figure 4: Ankyrin-G negatively regulates NF-κB reporter-gene activity.

(A) AnkG-eGFP inhibits neuronal constitutive NF-κB⁶¹ activity. Mature cortical neurons were transfected with ankG-eGFP or p65-eGFP and κB-dependent luciferase reporter activity was monitored in Dual-Luciferase assays (n = 13–19 wells pooled from 3 experiments, Kruskal-Wallis followed by Dunn’s test). (B) Efficient suppression of ankyrin-G expression at the axon initial segment following transduction with ankyrin-G-specific small-hairpin RNA. Mature cortical neurons were transfected with ankyrin-G shRNA or control vector together with eGFP for 5 days and immunolabeled. Scale bar, 10 μm. Note transfected ankyrin-G positive AIS marked by arrow or negative cell bodies marked by asterisk. One transfected cell is shown magnified in the boxed area. (C) Depletion of ankyrin-G expression in mature cortical neurons induces NF-κB activity. Mature cortical neurons were transfected with ankG- or p65-specific shRNA together with Dual-Luciferase reporter plasmids for 5 days and κB-dependent reporter gene expression was determined (n = 12 wells pooled from 2 experiments, Kruskal-Wallis, Dunn’s post-hoc test, experiment was repeated 2 times with equivalent results).