Western blotting

At the designated times, peritoneal cells (neutrophils) were harvested and lysed with cell lysis buffer containing 0.1% Triton X-100 and complete protease and phosphatase inhibitor cocktail as described [42]. Equal amounts of proteins were separated by SDS-PAGE, and transferred to a polyvinyl difluoride membrane. The nonspecific binding sites on the membrane were blocked with 5% non-fat dry milk in TBS supplemented with 0.1% Tween 20 for 1 h before proteins were allowed to react with specific primary antibodies against peptidylarginine deiminase-4 (PAD-4) (Cell Signaling), neutrophil elastase (Abcam), Gp91^phox (Abcam), P47^phox (Santa Cruz), P67^phox (Santa Cruz) and GAPDH (Cell Signaling) at 4°C overnight. The membrane was washed three times with TBS containing 0.1% Tween 20 (0.1% TBST) and incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. The immunoreactive bands in the membrane were detected by the chemiluminescence method (Amersham). Percent of total area was calculated for each band for PAD-4 using NIH ImageJ 1.51d software, and expressed relative to neutrophil elastase bands as the mean ± SE of three bands per group.