Linear ubiquitination is required for the formation of NEMO-containing structures in response to IL-1, but not TNF. (A) Control and HOIL1-deficient human fibroblasts were left untreated or stimulated with TNF or IL-1, as indicated. Cells were permeabilized with saponin before fixation and then processed for the detection of NEMO by immunofluorescence. Nuclei were stained with DAPI (blue). Bars, 10 µm. (B) The number of NEMO spots per cell in the experiment presented in A presented as mean values ± SD (error bars; n > 60 cells analyzed). Unpaired t tests were used to determine the significance of differences. ***, P < 0.001; **, P < 0.01. (C) NF-κB activation status in control and HOIL1-deficient cells stimulated with IL-1 and TNF for the times indicated was monitored by the detection of phosphorylated IKKα/β (p-IKK), IKKβ, and IκBα on immunoblots.