Figure 4. NF-κB and MAPK activity are preserved in patient MoDCs.

(A) Reduced levels of NEMO expression did not alter LPS-stimulated NF-κB and MAPK activity. Control and patient MoDCs were stimulated with LPS (2.5 μg/ml) for the indicated times; then proteins were extracted and subjected to Western blot analysis. Blots were probed with antibodies to NEMO, IKKα, IKKβ, phospho-IKKα/β (p-IKKα /β), IκBα, or GAPDH. Representative data from 2 independent experiments are shown. (B) Reduced NEMO expression does not affect NF-κB nuclear migration and DNA binding upon LPS stimulation. After LPS activation, MoDCs were fixed, permeabilized, and immunostained with anti-RelA antibody (left panels, red) or anti-cRel antibody (right panels, green) together with a nuclear-specific marker (blue). Original magnification, ×40. (C) Quantification of nuclear RelA and cRel staining. 15 images were selected for quantification. Data shown are mean ± SD. SI, stimulation index. (D) In cellular extracts prepared from LPS-activated (2.5 μg/ml) MoDCs, NF-κB DNA–binding activity was measured by ELISA (TransAM). One experiment of 2 is shown.