Figure 4.
Figure 4. NEMO-containing structures colocalize with the activated TNF receptor but not with the activated IL-1R. U2OS cells were incubated successively with biotinylated-TNF (A) or biotinylated-IL-1 (B) and avidin-A488 at 4°C. Cells were fixed directly or shifted to 37°C conditions for 5 min to allow stimulation. NEMO (red) and the activated TNF-R or IL-1R (avidin-A488, green) were detected by immunofluorescence. Competition experiments with unlabeled cytokines (in excess) were performed after the first incubation with biotinylated cytokines to assess probe specificity. The insets in the images show magnified areas (indicated by the boxed regions). Bars, 5 µm. (C) Quantification of the colocalization of NEMO and the activated TNF-R (blue diamonds) and activated IL-1R (red boxes) after 5 and 15 min of stimulation. Data are presented as mean values ± SD (error bars; n > 100 cells analyzed).

NEMO-containing structures colocalize with the activated TNF receptor but not with the activated IL-1R. U2OS cells were incubated successively with biotinylated-TNF (A) or biotinylated-IL-1 (B) and avidin-A488 at 4°C. Cells were fixed directly or shifted to 37°C conditions for 5 min to allow stimulation. NEMO (red) and the activated TNF-R or IL-1R (avidin-A488, green) were detected by immunofluorescence. Competition experiments with unlabeled cytokines (in excess) were performed after the first incubation with biotinylated cytokines to assess probe specificity. The insets in the images show magnified areas (indicated by the boxed regions). Bars, 5 µm. (C) Quantification of the colocalization of NEMO and the activated TNF-R (blue diamonds) and activated IL-1R (red boxes) after 5 and 15 min of stimulation. Data are presented as mean values ± SD (error bars; n > 100 cells analyzed).

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