Figure 1.

PAD2 and PAD4 are present in primary granules and degranulated extracellularly in response to soluble stimuli. (A) Neutrophil granules were isolated by sucrose gradient ultracentrifugation of neutrophil lysates (5 × 10⁷). Coomassie Blue stain of polyacrylamide gel loaded with neutrophil primary granules and secondary and tertiary granules. Western blot detection of PADs in respective fractions, including PAD6 (negative control), primary granule marker neutrophil elastase (NE), and secondary granule marker hCAP-18. Recombinant human proteins of PAD isotypes loaded (1.25 ng) to the left of the molecular weight marker (MWM) as positive controls. Representative images from n = 6 biological repeats. (B–E) Neutrophils (1.5 × 10⁷) were left unstimulated (un) or stimulated for 10 min with TNFα or TNFα and fMLP. Extracellular supernatants were assessed by Western blot for PAD2, PAD4, and the primary granule marker, NE. Neutrophil pellets were probed for β-actin (loading control). (C–E) Abundance of extracellular PADs was quantified by densitometric analysis of immunobands. Data are expressed as relative densitometry units (DU), with representative Western blots presented (N = 6 biological repeats, one-way ANOVA, followed by Tukey’s post-hoc multiple comparison test).