ARG1 is exocytosed by activated neutrophils. (A) Purified pb-PMNs were incubated for 15 minutes with or without the addition of 2.5 μg/mL PMA (phorbol myristate acetate), 100 nM fMLP (formyl-methionyl-leucyl-phenylalanine), or 50 ng/mL TNF α (tumor necrosis factor α). Supernatants containing granule proteins and pellets corresponding to equal amounts of cells were subjected to Western blot analysis (ARG1) or Western blot and ELISA analysis (MPO, LF, GEL). For Western blot analysis supernatants obtained from 3.75 × 106 stimulated cells as well as lysates of 3.75 × 106 stimulated cells were used. The percentage of MPO, LF, and GEL released by neutrophils was calculated as the amount of protein detected in the supernatant divided by the total amount of protein detected in the supernatant and pellet multiplied by 100. (B) Highly specific detection of ARG1 in gelatinase granules of neutrophils by Western blot analysis. A subcellular fractionation highly enriched in gelatinase granule (GG) was subjected to Western blot analysis using mouse anti–human ARG1 antibody. Prior blocking of the mouse anti–human ARG1 antibody by recombinant human ARG1 before probing of membranes reveals no cross-reactivity but a highly specific detection of only ARG1.
