FIGURE 3 RAW DATA MYD88 WT FULL LENGTH DILUTIONS SEEDED

Number of photons from time traces from approx 50 MYD88 dilutions with and without MYD88 cherry tagged seeds, raw data analysed in additional files corresponding to (A) Schematic diagram
of the principle of two-colour seeding experiments testing the self-replication
propensity of full-length MyD88 filaments. Full-length MyD88 is expressed in an mCherry-tagged version above its
supercritical concentration to create filaments, which are gently spun and
washed, then sonicated to increase the number of fragments. These “seeds” are
then mixed in a sample expressing GFP-tagged full-length MyD88 at sub-critical concentrations. (B) Example of fluorescence time
trace for MyD88 at 10 nM concentration. Unseeded sample demonstrating a
monomeric time trace profile (above) with the seeded sample (below) showing
polymerisation of GFP-MyD88 upon the addition of MyD88 ‘seeds’. (C) The B
parameter (brightness) correlating with number of oligomers detected in typical
time-traces as a function of protein concentration (nM), with and without
“seeds” introduced. The subcritical, supercritical and “meta-stable” zones are
labelled. Values are from approx. 50 repeated dilution experiments with the
various protein concentrations and corresponding brightness values obtained
plotted.