Figure 5: The transcription factor p65/NF-κB localizes to the axon initial segment.

(A–D) Mature cortical neurons were cytosol-extracted using a Triton-X100-based buffer. Extractions were performed at 4 °C. Then, neurons were either immunolabeled using a rabbit monoclonal anti-p65 (green, D14E12, CST), a rabbit monoclonal anti-cJun (green, Cell Signaling Technology) or a mouse monoclonal p65/NF-κB-specific antibody (green, pan-p65, F-6, SCBT) together with either mouse monoclonal (413) or rabbit polyclonal ankyrin-G-specific (H-215, both SCBT) antibodies. AIS highlighted by arrows. (D) Experiment was conducted as in C, with single AIS magnified. Note the labeling of pan-p65 immunoreactivities along the axon initial segment. Color overlay is depicted following background substraction. Scale bars, 10 μm. (E) p65-paGFP exhibits decreased mobility in the AIS. Following photo-activation of p65-paGFP or paGFP in DIV12–19 rat hippocampal neurons, fluorescence intensity decline was monitored. Representative time course images of photo-activated p65-paGFP or paGFP in the AIS are depicted. DsRed2 fluorescence (not shown) was used to identify the axon initial segment of transfected neurons based on morphological criteria. Times indicate time points after photo-activation. Regions marked with blue boxes were irradiated with 405 nm light to photo-activate the paGFP/p65-paGFP. Note that 1 second after photo-activation, p65-paGFP fluorescence increase is mainly confined to the irradiated region, while paGFP fluorescence has already spread over the region’s boundaries. Scale bars, 2 μm. (F–H) Traces of mean fluorescence intensity of p65-paGFP and paGFP in the irradiated region ±SEM, n = 3–4 experiments, per experiment 6–12 neurons were analyzed. (I) Bar graphs depict average time point of over 90% fluorescence intensity reduction after photo-activation in 28–33 neurons per group in 3–4 different platings. Time points were determined from data represented in the traces. Neurites of similar morphology and widths were imaged (F’–H’). No significant differences were found between paGFP and p65-paGFP expressing neurons. n.s.: not significant, one-way ANOVA.