Anykd mini protean tgx gels

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.

C2C12 cells were pre-treated with TubA for 24 h and then treated for 30 min with TGF-ß1. Cells were then collected and pelleted at 300 g at 4 °C for 3 min. Pellets were resuspended with 10 volumes of ice-cold E1 buffer (20 mM NaOH pH 7.6, 5 mM Potassium Acetate, 0.5 mM MgCl2) complemented with 1 x protease inhibitor cocktail and 1 x phosphatase inhibitor cocktail and placed in 1 mL Dounce homogenizer. Cells were homogenized with 25 strokes of the loose pestle and incubated 10 min on ice before centrifugation at 2100 × g at 4 °C for 3 min. The supernatant was collected (cytoplasm extract, CE) and the pellet was resuspended in the same volume of E1 buffer supplemented of 600 mM NaCl. The suspension was incubated 30 min on ice and centrifuged at 20,000 × g for 20 min. The supernatant was collected (nuclear extract, NE) and the pellet was resuspended in the same volume of E1 buffer supplemented of 600 mM NaCl and Benzonase (1/1000, Millipore, 71205) and correspond to the chromatin extract (Chrm). The same volume of each fraction was loaded on Any kD Mini PROTEAN TGX gels (Biorad), transferred on nitrocellulose membrane blotting and revealed with indicated antibody.

Approximately 0.4 mg/mL of protein in 20 mM Hepes pH 8.0, 10 mM sodium chloride was crosslinked using 20 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 20 mM N-hydroxysuccinimide (NHS) in 20 mM sodium phosphate, pH 7.0, 150 mM NaCl for various time points. The reaction was stopped by the addition of 0.5 M Tris-HCl pH 8.0 to a final concentration of 0.25 M. The products were analyzed on NuPAGE 3–8% Tris-Acetate gels (Life Technologies, Carlsabd, CA) or an Any kD Mini-PROTEAN TGX gels (BioRad) using a silver-staining kit (Thermo Scientific).