Nylon nitrocellulose membranes

Total protein was isolated with NucleoSpin TriPrep kit (Macherey-Nagel) from 3 × 10⁶ PMNs. Proteins from the nuclear and cytoplasmic fractions were isolated by using the Nuclear and Cytoplasmic Protein Extraction Kit (Thermo Scientific). Western blotting was performed by using AnykD Mini-PROTEAN TGX Gels (Biorad, Hercules, CA, USA) and nylon/nitrocellulose membranes (Biorad). Primary and secondary antibodies used were: rabbit anti-PAD4 (Abcam), rabbit anti-MPO (Cell Signalling Technologies, Beverly, MA, USA), mouse anti-β-Actin (Sigma-Aldrich), goat anti-Mouse and/or anti-Rabbit, human anti-HRP (Southern Biotech). HRP activity was detected by using SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific). Equal loading was verified by using beta-actin or histone H3, when appropriate. Western blots of citrullinated H3 (citH3) protein were performed according to Shechter et al.[25 (link)]. Densitometric analysis and protein quantification of the Western blots was performed by using the ImageJ software.

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 cellular protein was extracted using Pierce RIPA Buffer (Thermo Scientific) supplemented with PhosSTOP Protease Inhibitor Cocktail Tablets (Roche Life Science). For the placenta, frozen tissues were thawed for several minutes in pre-chilled RIPA buffer and homogenized with a Polytron (Kinematica). Western blotting was performed by using AnykD Mini-PROTEAN TGX Gels (Biorad, Hercules, CA, USA) and nylon/nitrocellulose membranes (Biorad). Primary and secondary antibodies used were: rabbit anti-IRS1 (1:500, sc-559, Santa Cruz Biotechnology), rabbit anti-GLUT4 (1:1,000, 07-1404, Millipore), mouse anti-β-Actin (1:5,000, Sigma-Aldrich), goat anti-Mouse (sc-2005, Santa Cruz Biotechnology), and/or anti-Rabbit (sc-2004, Santa Cruz Biotechnology), human anti-HRP (1:2,000, Santa Cruz Biotechnology). HRP activity was detected by using SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific). Densitometric analysis and protein quantification of the western blots was performed by using Image Lab Software (version 5.2.1). Blots were stripped briefly in 0.2 M sodium hydroxide and reprobed for mouse anti-β-Actin (Sigma-Aldrich) as a loading control.