Membrane-tethered IL1β triggers the relocalization of MyD88 to the cell surface and nuclear translocation of RelA. (A) The schematic of a SLB functionalized with IL1β labeled with mScarlet. (B) TIRF and brightfield microscopy images of EL4 cells expressing MyD88-GFP after landing on a SLB functionalized with IL1β-mScarlet. Clusters of IL1β-mScarlet formed at the cell–SLB interface. MyD88-GFP was recruited to clusters of IL1β-mScarlet. (C and D) EL4 cells were fixed (60 min after SLB contact) and stained for MyD88-GFP (green) and RelA (magenta), with DAPI staining of nuclei (blue). Cells were imaged with confocal microscopy. (C) Schematic shows the position of the confocal micrograph slice. Cells in contact with IL1β-functionalized SLBs relocalize RelA to the nucleus. (D) Reconstructed axial view of cells shown in C, showing the localization of MyD88 to the cell–SLB contact zone and RelA to cell nucleus under IL1β stimulation. (E) Quantification of RelA nuclear staining intensity. Bar represents mean ± SEM from n = 3 experimental replicates. Scatter plot symbols represent independent replicates, smaller gray symbols represent single-cell measurements, and superimposed larger symbols represent the averages from experimental replicates. fluo., fluorescence.