Mn protein quantification assay

For luciferase assays, approximately 24 hrs after transfection, cells were lysed in a 100 μl Luciferase Assay Tropix Lysis solution (ThermoFisher Scientific), with 0.5 mM DTT. Cells were scraped, transferred to Eppendorf tubes and then centrifuged for 3 minutes at 12,000 g. Twenty μl of the supernatant was used in a 96-well microtiter plate, and a 50 μl luciferase buffer (Promega, Madison, WI, USA) was added. A Luminometer (Labsystem, Luminoscan RT) used to measure lights units. Each experiment was repeated three times with three independent parallels for each experiment, and luciferase values were corrected for protein content in each sample. The total protein concentration was measured using the MN protein quantification assay (Macherey-Nagel GmbH, Düren, Germany).

Western blot analysis was performed as previously described [77 (link)]. Briefly, total protein was isolated by NucleoSpin TriPrep kit (Macherey-Nagel, Düren, Germany) from 5 × 10⁶ PMNs. All protein concentrations were determined with the MN Protein Quantification Assay (Macherey-Nagel, Düren, Germany). Western blotting was performed utilizing AnykD Mini-PROTEAN TGX Gels (Biorad, Hercules, CA, USA) and nylon/nitrocellulose membranes (Biorad, Hercules, CA, USA). Equal loading was verified using beta-actin. Western blots of citrullinated H3 (citH3) protein were prepared as described previously [81 (link)]. Gel documentation, densitometric analysis, and protein quantification of the Western blots was performed using the ChemiDoc XRS+ maging system (Biorad, Hercules, CA, USA) with the ImageLab 4.1 image analysis software (Biorad, Hercules, CA, USA).

Total protein was isolated by NucleoSpin TriPrep kit (Macherey-Nagel) from 5 × 10⁶ neutrophils. All protein concentrations were determined with the MN Protein Quantification Assay (Macherey-Nagel). Western blotting was performed utilizing AnykD Mini-PROTEAN TGX Gels (Biorad) and nylon/nitrocellulose membranes (Biorad). Primary and secondary antibodies utilized were rabbit anti-MPO (Cell Signalling Technologies), rabbit anti-PAD4 (Abcam), rabbit anti-citH3 (Abcam), mouse anti-β-Actin (Sigma), anti-rabbit HRP (Santa Cruz), and anti-mouse HRP (Santa Cruz). HRP activity was detected by using SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific). Equal loading was verified using beta-actin. Western blots of citrullinated H3 (citH3) protein were prepared as described previously (34 (link)). Gel documentation, densitometric analysis, and protein quantification of the western blots was performed using the ChemiDoc XRS+ imaging system (Biorad) with the ImageLab 4.1 image analysis software (Biorad).