Figure 3

Post-translational histone modifications of neutrophil extracellular traps (NETs) derived from primary human neutrophils. Primary human neutrophils were isolated from peripheral blood, immediately stimulated with 10 mM hydrogen peroxide for 4 hours, and NETs were harvested. NETs or the corresponding unstimulated neutrophils were assayed by MABA. A panel of anti-histone antibody epitopes (top) is shown, with PTMs according to the PTM key. A more limited PTM panel was selected on the basis of available epitopes not subject to proteolysis by considering those enriched or depleted during NETosis of neutrophils derived from human and murine cell lines (Figure 4 and Additional file 1: Supplemental Figure 4). Bottom: asterisks or carats indicate increase or decrease (respectively) of band intensity for indicated lane in NETs compared to unstimulated neutrophils. Band migration is shown for 10-15 kDa range and film exposure time was 20'. MABA, Multiple Antigen Blot Assay; PTM, post translational modification.