Ab176843

Antibodies for detecting citrullinated histone H3 (R2 citrullination, Ab176843 and R2-8-17 citrullination, Ab5103), total histone H3 (Ab24834), NCF1, NCF2, and NCF4 were from Abcam (Cambridge, MA). Antibodies for detecting PAD4 (MABE254) and citrullination (F95) were from EMD Millipore (Billerica, MA). Antibodies for PAD IP were developed as previously described^{40 (link)}. Recombinant human PAD2 and recombinant human PAD4 were generated in-house. Recombinant human histone H3, pan PAD inhibitor BB-Cl-amidine, and PAD4 inhibitor GSK484 were from Cayman Chemicals (Ann Arbor, MI). The cell fraction isolation kit was from Cell Signaling (Danvers, MA). Protein G beads, DPI, ionomycin, PMA, Hoechst 33342, HRP-labeled anti-mouse immunoglobulin G (IgG), and anti-rabbit IgG secondary antibodies were from Invitrogen (Carlsbad, CA).

An anti-histone H3 antibody was obtained from Cell Signaling Technology (#4499, MA, USA). An antibody against citrullinated histone H3 at R2 was purchased from Abcam (ab176843, Cambridge, UK). A secondary antibody conjugated with horse radish peroxidase (HRP) was obtained from Santa Cruz Biotechnologies (Dallas, TX, USA). The following reagents were used in this study: SYTOX Green and YO-PRO-1 (Thermo Fisher Scientific, MA, USA), MitoRed and Hoechst 33342 (Dojindo Laboratories, Kumamoto, Japan), A23187 and ionomycin (Wako Pure Chemical, Osaka, Japan), MNase (New England BioLabs, MA, USA), and RNase A (Sigma-Aldrich, MO, USA).

Proteins were separated by SDS–PAGE and transferred to nitrocellulose membrane using wet transfer. Membranes were blocked in 5% BSA in TBS containing 0.1% Tween-20 for 1 h at room temperature. Proteins were detected using primary antibodies against anti-H3 (Abcam ab10799, 1:1,000), anti-H3CitR2 (Abcam ab176843, 1:2,000), anti-NPM1 (Abcam ab37659, 1:200), and anti-GST (Abcam ab19256, 1:1,000) overnight at 4 °C and in secondary antibody at 1:5,000 for 1 h at room temperature. Membranes were incubated in Pierce ECL reagent and imaged using ImageQuant LAS 4000 (GE). Citrulline-containing proteins were modified on the membrane and detected using the antimodified citrulline detection kit (Millipore, 17-347) according to manufacturer’s instructions.