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.