Figure 1: MAL forms large assemblies in vitro and in vivo, TLR4TIR and MALTIR form co-assemblies, and MALTIR induces MyD88TIR assembly formation.
a, Negative-stain EM images of MALTIR and MALFL incubated at 30°C for 1 h. b, Negative-stain EM images of TLR4TIR and TLR4TIR +MALTIR incubated at equimolar concentrations, at 30°C for 1 h. c-d, SDS-PAGE analyses of the insoluble fraction after incubation of MALTIR, TLR4TIR, and MALTIR + TLR4TIR at 30°C for 1 h with each protein at 60 µM (c) or 30 µM (d). The results are typical of three experiments. e, Negative-stain EM images of MyD88TIR (60 µM), and MALTIR (6 µM) + MyD88TIR (60 µM) incubated at 30°C for 1 h. f, SDS-PAGE analyses of the soluble (SF) and insoluble (IF) fractions after incubation of MyD88TIR (60 µM), and MALTIR (6 µM) + MyD88TIR (60 µM) at 30°C for 1 h. The results are typical of three experiments. g-h, Turbidity assays of MyD88TIR (60 µM) assembly formation in the presence of sub-stoichiometric amounts of MALTIR (g), TRAMTIR and TLR4TIR (h). Similar trends were observed in three experiments. The magenta (MyD88-TRAM 20:1) and orange (MyD88-TLR4 10:1) lines are mostly hidden under the light blue line (MyD88-TLR4 20:1). i, Clustering of MyD88FL in HEK293 cells induced by MAL. Cells were transfected with plasmids expressing V5-tagged MyD88FL or MyD88TIR alone or in the presence of Myc-tagged MALFL or MALTIR. Cells were immuno-stained with anti-V5 and anti-Myc antibodies and analyzed by flow cytometry. Cells with low MyD88-V5 expression were selected (Supplementary Fig. 4) for analysis of fluorescent pulse-height vs. area for MyD88-V5. Cells with clustered MyD88 have an elevated height-to-area ratio (boxed). The results are typical of three experiments. The original images of gels in (c), (d) and (f) can be found in Supplementary Data Set 1.