Aernoud Fiolet

276 Chapter 11 Table 2. Distribution of NLRP3 protein in density sub-fractions of the TEX precipitate after density gradient centrifugation. TEXDensity Gradient Subfractions NLRP3 Protein in pg/mL Density in g/mL TEX 1 Not detectable 1.02 TEX 2 0.74 1.02 TEX 3 9.06 1.03 TEX 4 18.3 1.04 TEX 5 15.1 1.06 TEX 6 6.3 1.11 TEX 7 2.9 1.12 TEX 8 4.49 1.13 TEX 9 1.41 1.16 TEX 10 Not detectable 1.32 Highest concentrations of NLRP3 protein were observed in the 3-6th subfraction. Western Blot shows highest levels of CD9, which is an EV specific protein, in the same subfractions (Figure 3). These results show that NLRP3 is indeed expressed in EVs. Abbreviations: EV, extracellular vesicle; IL-6, interleukin-6; NLRP3, nucleotide-binding oligomerization domain-, leucine-rich repeat-, and pyrin domain-containing protein 3 Extracellular vesicle NLRP3 protein level Iodixanol density gradient centrifugation showed highest EV NLRP3 protein levels in the 3rd to 6th subfraction of this density gradient with the density of 1.03-1.11 g/ml. Subfractions with this density also contain the highest CD9 levels (Figure 3), and thereby indicate that NLRP3 is expressed in EVs (Table 2). Figure 3. Western Blot showing the distribution of CD9 Level in TEX subfractions. Subfractions 3-6 show highest levels of CD9. These fractions correspond with the density 1.03-1.11 (Table 2).