Figure 6: p65 increases ank3 promoter-dependent reporter-gene activity.

(A) Schematic drawing of a 2 kB stretch upstream of the putative transcription start site (TSS) of the mouse ank3 gene containing multiple putative NF-κB transcription factor binding sequences. (B) p65/NF-κB binds to the ank3 gene promoter. p65 ChIP following 2 h TNF-α (100 ng/ml) in mouse BV2 cells, primer numbers surrounding putative NF-κB sequences labeled as in (A). (C) qPCR of ank3 transcripts in PC12 cells following depletion of endogenous p65 protein. PC12 cells were transfected using p65-shRNA or ank3-eGFP vectors and analyzed. Fold-induction of ank3 transcripts was determined using 18S rRNA-specific primers for normalization (n = 3). (D) The ank3 promoter sequence highlighted in (A) was cloned into a pGL3-luciferase reporter vector and used in Dual-Luciferase-assays following transfection of PC12 cells (n = 8–16, Kruskal-Wallis test, Dunn’s post-hoc, p = 0.0094). (E) TSS proximal promoters bind p65. Proximal and distal parts of above sequence were similarly generated and transfected into mature cortical neurons with IKKβ and p65 expression vectors (n = 4, Mann-Whitney test within fragments, p ≤ 0.05 (proximal and distal)). (F) Similarly, p65 induces the ank3 promoter in primary cortical neurons. These were transfected with p65 and IκBα vectors together with the ankyrin-3-reporter (n = 6–8 wells, Kruskal-Wallis followed by Dunn’s, p = 0.0003). (G) Depletion of endogenous p65 suppresses ankyrin-3-reporter activity. Cortical neurons were transfected using control-/p65-shRNA constructs together with the ank3-reporter construct for 5 days, relative luciferase activities were determined (n = 6 wells, Mann-Whitney test, p = 0.0022). (H) Overexpression of serine536-phospho-mimetic p65-mutants (S536D) results in higher ankyrin-G expression at the AIS than wild-type p65. Ankyrin-G immunofluorescence along the AIS was determined following three days of transfection. (I) Quantification of the AUC determined from the single traces obtained from the p65 mutants in ((H) n = 19–33 axons, Kruskal-Wallis test, Dunn’s post-hoc *p = 0.0093). (J) Mature cortical neurons were transfected using the ank3-promoter in conjunction with ankG-eGFP and lysed after two days. Relative ank3-luciferase activity was determined (n = 8 wells, parametric data analyzed by two-sided t-test, p = 0.00315).