NEMO and phosphorylated IKKs concentrate into punctate structures in response to cytokine stimulation. (A) U20S cells left untreated or treated with IL-1 (12 min) or TNF (5 min) were subjected to saponin extraction before fixation and processed for the detection of NEMO (green) and phospho-IKKs (p-IKK, red) by immunofluorescence. Nuclei were stained with DAPI (blue). The insets in the images show magnified areas (indicated by the boxed regions). Bars, 5 µm. (B and C) The number of NEMO foci (blue diamonds) and pIKK-positive NEMO foci (red circles) per cell were quantified after IL-1 (B) and TNF (C) stimulation. Data are presented as mean values ± SD (error bars; n > 40 cells analyzed).