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. Author manuscript; available in PMC: 2017 Jul 14.
Published in final edited form as: Sci Transl Med. 2016 Dec 14;8(369):369ra176. doi: 10.1126/scitranslmed.aaj1921

Fig. 3. The pore-forming toxin of Aa induces cellular hypercitrullination in neutrophils.

Fig. 3

(A) Human neutrophils (PMNs) and Aa HK1651 cells were incubated alone or co-incubated in the presence or absence of a blocking antibody against LtxA (αLtxA) for 1 h at 37°C. Total citrullinated protein (AMC), citrullinated H3 (citH3), and histone H3 (H3) (loading control) were detected by immunoblotting. (B) PMNs and PBMCs were incubated with increasing concentrations of purified LtxA (0-1.0 μg/mL) for 1 h at 37°C. AMC and citH3 by immunoblotting are shown. H3 and PAD4 were visualized to demonstrate loading. (C) PMNs were incubated with purified LtxA (0.3 μg/mL) at 37°C for 0-60 min. PMNs incubated with LtxA + 5 mM EGTA for 60 min were used as controls. AMC and proteinase 3 (PR3) (loading control) were detected by immunoblotting. (D) Total citrullinated protein at each time point in C was quantified by densitometry (mean optical density ±SEM). Data are from two independent experiments. (E) Human monocyte-derived macrophages were incubated with increasing amounts of purified LtxA (0-1.0 μg/mL) for 3 h at 37°C. AMC and H3 (loading control) by immunoblotting are shown. The experiments in A, B, and E were performed on at least 3 separated occasions, with similar results.