Figure 3.

Multiple IκBα properties contribute distinct characteristics to NFκB control. Nuclear localization of IκBα is required for the termination of NFκB activity (a,b). Nuclear export function of IκBα is required for post-repression activation of NFκB activity (c,d). IκBα protein half-life control is critical for sustained NFκB dynamics (e,f). IκBα^−/− MEFs were reconstituted with NFκB-inducible wild-type or NLS mutant (a,b), NES mutant (c,d) or the 5M mutant (e,f) form of IκBα. The cells were treated with 1 ng ml⁻¹ of TNF and nuclear extracts were analysed by EMSA for NFκB activity and corresponding cytoplasmic extracts subjected to western blotting with indicated antibodies (a,c,e). Bar graphs show quantification of EMSA; curves are modelling the result of NFκB activity in single cells upon stimulation with 10 ng ml⁻¹ of TNF. Real-time fluorescent images of IκBα^−/−β^−/−ε^−/−RelA^−/− MEFs reconstituted with AcGFP1-RelA and NFκB-inducible IκBα NLS mutant (b), NES mutant (d) or the 5M mutant (f) IκBα (showing cellular localization of RelA at indicated time points). Below the fluorescent images, single-cell traces show the ratio of nuclear to cytoplasmic localization of AcGFP1-RelA in fluorescent images (left) as well as the average curve and standard deviation of the single-cell traces (right).