Molecular characterization of NF-κB/IκBα dynamics in IκBαM/M KI neurons. A) Diagram depicting the IκBα autoinhibitory loop and the effect of our KI mutations. B) Representative Western blots of IκBα protein levels in cytoplasmic fractions from WT and KI primary neurons treated with TNFα (top) or IL-1β (bottom) for the indicated length of time. C) Quantification of Western blots of IκBα protein levels following TNFα stimulation normalized to the loading control and to baseline expression. N = 2-5 cultures each, * = p < 0.05, ** = p < 0.01. D) Expression of IκBα mRNA was measured by quantitative RT-PCR in WT and KI primary neurons before and after TNFα stimulation for 1 hr and levels were normalized to GAPDH expression and then to WT unstimulated levels. N = 3 cultures each, *** = p < 0.001. E) Representative EMSAs of NF-κB activity in nuclear fractions from WT and KI primary neurons treated with TNFα (top) or IL-1β (bottom) for the indicated length of time. Specificity of the shifted band is confirmed through the addition of either excess unlabeled consensus (WT) or mutant (MT) probe in the last two lanes. Free labeled probe (F) alone was run in the first lane. F) Quantification of NF-κB activity from nuclear fractions following TNFα stimulation using an ELISA-based assay specific for the p65 subunit. N = 3-4 cultures each, *** = p < 0.001.