Myddosomes are highly stable molecular assemblies that do not exchange with uncomplexed MyD88, IRAK4, or IRAK1. (A) TIRF images of MyD88-GFP before and after (0 and 30 s) photobleaching. The red box indicates the position of the Myddosomes on which the FRAP beam was focused. The blue box indicates unbleached control. Right: Cropped time series of the photobleached and control Myddosomes. (B and C) TIRF images of MyD88-GFP/IRAK4-mScarlet– (B) and MyD88-GFP/IRAK4-mScarlet–(C) expressing cells before photobleaching. The green and magenta boxes indicates the position of the IRAK4-mScarlet– and IRAK1-mScarlet–labeled Myddosome on which the FRAP beam was focused, respectively. The blue box indicates unbleached control. Right: Cropped time series of the photobleached and control IRAK4-mScarlet– (B) or IRAK1-mScarlet– (C) labeled Myddosome. (D–F) Normalized recovery curves for MyD88 (n = 61 cells), IRAK4 (n = 22 cells), and IRAK1 (n = 53 cells). All yielded a maximal recovery of <20% during the 60 s of observation.