Roti load 2

IL-1β serum levels were determined by high sensitive IL-1ß Kit (Thermo Fisher Scientific), anti-MPO-antibodies (ORG 519 Orgentec), anti-PR3-antibodies (ORG 618, Orgentec) by ELISA, and FHR1 concentration by Human CFHR1 ELISA Kit (RayBiotech) according to the protocol provided by the manufacturers. CRP levels were measured by module C701 (Cobas8000). All samples below CRP detection limit of 5 mg dL⁻¹ were defined as 5 mg dL⁻¹. Presence of FHR1 in NHS samples was determined by Western Blot analysis and absence confirmed by PCR^{19 (link)}. Roti®-Load 2 (2.5 μL, Roth) was added to NHS (1 μL) diluted in DPBS (10 μL) and samples were separated by SDS-PAGE. Immunoblotting was performed with polyclonal FH antiserum (1:7.500, CompTech, LOT #4) and HRP-conjugated goat antibody (1:2.500, Dako, LOT 00073984).

Eluted EVs were lysed in 10X cell lysis buffer (Cell Signaling Technology, Germany) with protease inhibitor cocktail B (Santa Cruz, United States of America) and were mixed with Roti-Load 2 (Carl Roth, Germany). For reducing conditions DTT was added. 24 μl of the sample was loaded on a 10% or 12% SDS-polyacrylamide electrophoresis gel and transferred to a nitrocellulose membrane. Subsequently, it was blocked in 5% non-fat dry milk and incubated with anti-CD63 (Invitrogen, Germany) and anti-CD81 (Invitrogen, Germany) under non-reducing conditions, anti-Flotillin-1 (BD Biosciences, United States of America) and anti-Calnexin (Cell Signaling Technology, United States of America) under reducing conditions. Afterwards, the membrane was thoroughly washed and incubated with the secondary antibody coupled with a horseradish peroxidase. The chemiluminescence was detected using the ECL Plus Western Blotting Detection Reagent (Amersham, United States of America) on the Luminescent Image Analyzer LAS-3000 (FUJIFILM, Japan).