Rabbit anti pad4

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.

The 5 × 10⁴ isolated PMNs were seeded on poly-L-lysine-coated glass coverslips (BD Biosciences, San Jose, CA, USA) in tissue-culture wells and allowed to settle before stimulation, as described earlier. Coverslips were rinsed with ice-cold HBSS and the cells fixed with 4% paraformaldehyde and blocked overnight (HBSS with 10% goat serum, 1% BSA, 0.1% Tween20, and 2 mM EDTA) at 4°C. NETs were detected with rabbit anti-NE (Abcam, Cambridge, MA, USA), rabbit anti-MPO (Dako, Glostrup, Denmark), two different rabbit anti-PAD 4 (Abcam), mouse anti-PAD4 (Abcam), mouse anti-histone H1 + core proteins (EMD Millipore, Billerica, MA, USA), and rabbit anti-citrullinated histone H3 (citH3, Abcam). Secondary antibodies were goat anti-rabbit IgG AF555, goat anti-rabbit IgG AF488 (Invitrogen Life Technologies, San Diego, CA, USA), and goat anti-mouse IgG AF647. DNA was stained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich), and NETs were visualized by using a Zeiss Axioplan 2 Imaging fluorescence microscope in conjunction with a Zeiss AxioCam MRm monochromatic CCD camera and analyzed with Axiovision 4.8.2 software (Carl Zeiss). A minimum of 20 fields (at least 1,000 PMNs) per case was evaluated for MPO/NE and DNA co-staining; nuclear phenotypes and NETs were counted and expressed as percentage of the total number of cells in the fields.

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).