Mmp 9 2c3 sc 21733

Proteins were extracted from 1.5×10⁷ neutrophils suspended in 120μl of lysis buffer (50mM Tris-HCl pH=7.5, 1% SDS, 10% (v/v) protease inhibitors (p8340, Sigma-21 Aldrich, MO), 1% Triton X100, 150mM NaCl) and incubated for 30 min on ice. Protein concentrations were determined for each sample with Bradford assay (BioRad), and equal protein loads were supplemented with 5× Laemmle sample buffer for a final concentration of 1× and boiled for 10 min at 100 °C. Total cell protein extracts were separated over 12 or 15% SDS-PAGE and transferred to a nitrocellulose membrane (Whatman). Membranes were blocked with 5% BSA, incubated with specific antibodies against human PAD4 (ab96758; Abcam), Anti-Neutrophil Elastase (G-2; SC-55549; Santa Cruz Biotechnology, Inc.), Anti-MPO/Myeloperoxidase (266-6K1; SC-52707; Santa Cruz Biotechnology, Inc.), MMP-9 (2C3; SC-21733; Santa Cruz Biotechnology, Inc.), and the housekeeping protein GAPDH (Cell Signaling 2118S) as a loading control. Antibodies were diluted in TBST 1% BSA and incubated for 1 h, followed by incubation with secondary antibody conjugated to horse reddish peroxidase (HRP) (Jackson). Signals were visualized in G-box (Syngene) (Additional file 2).

Proteins were extracted from 1.5×10⁷ neutrophils suspended in 120μl of lysis buffer (50mM Tris-HCl pH=7.5, 1% SDS, 10% (v/v) protease inhibitors (p8340, Sigma-21 Aldrich, MO), 1% Triton X100, 150mM NaCl) and incubated for 30 min on ice. Protein concentrations were determined for each sample with Bradford assay (BioRad), and equal protein loads were supplemented with 5× Laemmle sample buffer for a final concentration of 1× and boiled for 10 min at 100 °C. Total cell protein extracts were separated over 12 or 15% SDS-PAGE and transferred to a nitrocellulose membrane (Whatman). Membranes were blocked with 5% BSA, incubated with specific antibodies against human PAD4 (ab96758; Abcam), Anti-Neutrophil Elastase (G-2; SC-55549; Santa Cruz Biotechnology, Inc.), Anti-MPO/Myeloperoxidase (266-6K1; SC-52707; Santa Cruz Biotechnology, Inc.), MMP-9 (2C3; SC-21733; Santa Cruz Biotechnology, Inc.), and the housekeeping protein GAPDH (Cell Signaling 2118S) as a loading control. Antibodies were diluted in TBST 1% BSA and incubated for 1 h, followed by incubation with secondary antibody conjugated to horse reddish peroxidase (HRP) (Jackson). Signals were visualized in G-box (Syngene) (Additional file 2).