Ready made gels

Subcellular protein fractions were isolated using a ready-made kit following the instructions by the manufacturer (ThermoFisher Scientific). Granulocyte proteins and proteins of enzymatically treated isolated nuclei were isolated after adding HALT protease inhibitor (ThermoFisher Scientific) directly to the culture well and subsequent prompt heat denaturation at 95°C for 5 min in LDS gel loading buffer. Protein quantification was performed using Bradford reagent (Carl Roth). For further analysis, Western blots were performed after SDS-PAGE using ready-made gels (Bio-Rad). Digital image acquisition was performed using a Bio-Rad ChemiDoc Imaging System (Bio-Rad). Images of molecular size markers and specific Western Blots were merged using the manufacturer's recommended software. Nitrocellulose membranes of western blots were reprobed after washing in Tris buffered saline, 0.1% Tween-20 (TBS/T) and stripping in Tris-HCl pH 6.8, SDS and β-mercaptoethanol.

Intracellular MMP‐9 protein contents in PF were determined by immunoblot. Day 7 PF plated in T‐25 tissue culture flasks were treated with 0.1–100 ng/mL MCP‐1 (Thermo Scientific, Rockford, IL) overnight or were treated with buffer alone. Following treatment, cells were scraped in 4°C PBS, and obtained protein lysates were resuspended in Laemmli sample buffer, sonicated, heat‐denatured, and electrophoresed in 7–15% tris HCl Ready Made gels (Biorad, Hercules, CA). Resolved proteins were transferred to a PVDF membrane (Immobilon/Millipore, Bedford, MA). Membranes were blocked using Odyssey Blocking Buffer (Li‐Cor Biotechnology) and incubated with a rabbit monoclonal antibody against MMP‐9 (1:1000) (Abcam, Cambridge, MA) followed by a 680 IRDYE goat anti‐rabbit secondary antibody (1:8000) (Li‐Cor Biotechnology) and imaged using the Odyssey Imaging System fluorescence scanner (Li‐Cor Biotechnology). After restoring the membranes, they were incubated with a commercial mouse monoclonal antibody against beta actin (1:5000) followed by 800 IRDYE goat anti‐mouse (1:8000) (Li‐Cor Biotechnology). In a separate set of experiments, Day 7 PF were treated with BAPTA/AM (10 μmol/L), MCP‐1 (10 ng/mL), BAPTA/AM + MCP‐1, or ionomycin (10 μmol/L) (Calbiochem, San Diego, CA) for 10 min before harvesting cells and following the protocol above.