(a)
(b)
(c)
Figure 1: Neutrophil hypercitrullination can be induced by cell membrane lytic conditions. (a) Freshly isolated neutrophils cultured in medium with 2 mM CaCl2 alone or in presence of 1 μM ionomycin for 1 or 4 hours. Citrullinated proteins were detected by anti-citrulline antibody clone F95 (left panel) or anti-modified citrulline antibody (AMC) (right panel). Lower panel, immunoblot of the same filter with anti-β-actin as a loading control. (b) Freshly isolated neutrophils cultured in medium with 2 mM CaCl2 alone or with 1 μM ionomycin, 0.005% Triton-X, or 500 ng/mL perforin for 4 hours and immunoblotted with clone F95 antibody. Lower panel, immunoblot with anti-β-actin as a loading control. Similar results were obtained with neutrophils from four additional donors. (c) Densitometric scan of lanes 3, 4, and 6 of the blot in panel (b).