2.5 Immunoblotting
For Western blot analysis, the reaction was stopped by adding to the cell suspension an equal volume of 2x reducing Laemmli’s lysis buffer, added with 2 mmol/L sodium orthovanadate, 5 mmol/L EGTA, 5 mmol/L EDTA, 10 mmol/L sodium pyrophosphate, 10 mmol/L iodoacetic acid, 1 mmol/L phenylmethylsulphonyl fluoride, 10 mmol/L sodium fluoride, 10 µg/mL leupeptin and aprotinin, 1 mg/mL trypsin/chymotrypsin inhibitor. Samples were boiled for 10 min and centrifuged for 10 min at 10,000g. Aliquots of 100 µl, corresponding to 0.2 x 106 neutrophil total lysate (supernatant and cells), were loaded into 10% gradient sodium dodecyl sulphate-polyacrylamide gel. Proteins were transferred onto nitrocellulose sheets and nonspecific sites blocked using 1% bovine serum albumin (BSA) in Tris-buffered saline overnight at room temperature on a horizontal shaker. The presence of citrullinated Hystone-3 was analysed by immunoblotting with a rabbit, polyclonal, anti-Histone H3 (citrulline R2+R8+R17) (ab5103Abcam, Cambridge, Mass., USA), (1:1000; 1 hour at room temperature) specific antibody, followed by incubation with anti-rabbit ECL-conjugated secondary antibody (1:5000; 1 hour at room temperature). ECL reagent (Perkin/Elmer, Inc) (MA/USA) was used for the detection of luminescence, by UVITEK.