Additional file 1 of Low disease activity of microscopic polyangiitis in patients with anti-myosin light chain 6 antibody that disrupts actin rearrangement necessary for neutrophil extracellular trap formation

Additional file 1: Figure S1. Chronological G- and F-actin distribution in neutrophils stimulated by PMA. NETs were induced in peripheral blood neutrophils by 20 nM PMA. Before and 30 min, 1 h, and 3 h after incubation, the samples were fixed with 4% paraformaldehyde for 15 min and permeated with 0.5% Triton X-100 for 5 min at RT. Thereafter, the samples were reacted with 1:100 dilution of anti-β-actin mAb (mouse IgG1) at 4°C overnight. After rinsing with PBS, the samples were next reacted with 4 μg/mL Alexa Fluor 488-conjugated goat anti-mouse IgG1 antibody and 100 nM Acti-stain 555 phalloidin for 1 h at RT in the dark. The samples were finally mounted with the mounting solution containing DAPI. Anti-β-actin antibody recognizes G-actin, whereas phalloidin binds specifically to F-actin. Bar, 50 μm.