Figure 2. Phospho-P65-NF-κB expression in spinal cord neurons and NF-κB-TDP-43 interaction. To assess the level of inhibition of p65-NF-κB activation, achieved due to the super-repression, and compare them with that in ALS mice models, spinal cord sections were stained with neuron-specific NeuN antibody and phopho-p65 NF-κB antibody. Nuclei were stained with DAPI. Single-channel images from IκB-SR;TDP-43A315T (Ai-Aiii), TDP-43A315T (Bi-Biii), IκB-SR;TDP-43G348C (Ci-Ciii), TDP-43G348C (Di-Diii), IκB-SR (Ei-Eiii), and WT (Fi-Fiii) mice were analyzed separately using Fiji in a three step process. First, three corresponding single-channel images (NeuN, p65-NF-κB, DAPI) of every field were opened and stacked together. At the second step, neuronal boundaries were drawn (based on NeuN staining) using the free-hand selection tool, and the area was measured. Only neurons with an area > 250 µm2 were considered for this study. The nuclear boundary was also marked similarly (based on DAPI). Finally, the p65-NF-κB signal in the area marked as nuclear was measured and corrected for background fluorescence. For representational purposes, all channels in F are showing original boundary markings. Neurons from at least six sections per mouse with 3 mice in each group were considered. Original magnification ×20. The highlighted sections in each single channel images were cropped using Adobe Photoshop CS5, enlarged 300-fold, merged, and displayed in actual colors (Aiv, Biv, Civ, Div, Eiv, Fiv). Scale bar, 100 µm. Data were tabulated and the mean ± SEM plotted. ANOVA was performed using Kruskal-Wallis test followed by comparison between groups using Dunn's post-test. G, Adjusted p values. The neuronal nuclear phospho-p65 NF-κB expression was found to be significantly elevated in both the ALS mice model compared with IκB-SR and WT. Neurons in the spinal cord of double-transgenic mice (IκB-SR;TDP-43 mutants) showed marked reduction in presence of phospho-p65 NF-κB compared with respective TDP-43 mutants only. To assess whether the inhibition of p65-NF-κB release from the inhibitory κB complex affected its binding with TDP-43, spinal cord lysates from double-transgenic mice (IκB-SR;TDP-43 mutant) and its littermate TDP-43 mutant and IκB-SR were immunoprecipitated with anti-P65-NF-κB antibody and probed with anti-hTDP-43 antibody. Results showed a clear reduction in binding between the two proteins due to IκBα super-repression (dotted arrow). No interaction was expectedly observed in IκB-SR mice, which lacked the human TDP-43 expression. H, Representative of three independent experiments; Extended data Figure 2-2.