Histone h3 antibody

Neutrophils were stimulated as described in “neutrophil oxidative burst” for 5-10 min at 37°C. Cell lysates were prepared using boiling SDS lysis buffer (2% SDS, 62.5 mM Tris, pH 6.8, 5% β-mercaptoethanol, and 10% glycerol) supplemented with phosphatase and proteinase cocktail inhibitors (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Protein concentration was determined by Pierce^TM BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Equal amounts of proteins were separated on 10% SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blotted with p-MEK-1 (B-4 for pSer298, #sc-271914), p-ERK 1/2 (pT202/pY204.22A, #sc-136521), or p-IκBα (39A1431, #sc-52943) antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) to determine the activation of MEK, ERK, and NF-êB. The levels of citrullinated histone H3 was determined by western blot analysis with anti-citrullinated H3 antibody (citrulline R2 + R8 + R17, #ab5103) from Abcam (Cambridge, MA, USA) using the similar procedure except that neutrophils were stimulated for 2 hr before processed for western blot. And Western blot analysis with anti-β-actin antibody (#4967, Cell Signaling Technology, Danvers, MA, USA) and histone H3 antibody (#MA5-15150, Thermo Fisher Scientific) served as loading controls. Intensity of protein bands in western blot was quantified by ImageJ.