*4.7. Immunoprecipitation of Citrullinated Protein*

The nuclear extract from macrophages stimulated by LPS was immunoprecipited by ACPAs-conjugated protein G Sepharose beads (Protein G Immunoprecipitaton kits, Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer's instruction. Then, protein molecules in the supernatant (immunoprecipitates) were then analyzed by Western blotting using anti-p65 antibodies as a probe.

#### *4.8. Western Blotting Analysis*

Cell lysate was electrophoresed and transferred to a polyvinyllidene difluoride (PVDF) sheet (Sigma-Aldrich, St. Louis, MO, USA). The membranes were nonspecifically blocked in a 1% skim milk solution and incubated with primary antibodies for PADI2 (ab56928), PADI4 (ab128086), and citrullinated histone 3 (cit-H3; ab5103), caspase-2 (ab179520) (Abcam, Cambridge, UK), caspase-3 (9662), cleaved caspase-3 (9661), caspase-8 (9746), cleaved caspase-8 (9496), caspase-9 (9502), cleaved caspase-9 (7237), p21-activated kinase 1 (PAK1) (2602), focal adhesion kinase (FAK) (3285), phospho-FAK(8556), paxillin (12065), phospho-paxillin (2541), p65 (8242), and heat shock protein 90 (hsp90) (4874) (Cell Signaling Technology, Danvers, MA, USA) followed by the respective HRP-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Blotting was visualized by chemiluminescence reaction (ECL; GE Healthcare, Little Chalfont, UK). The respective band intensities were measured using ImageJ (version 1.42; http://rsb.info.nih.gov/ij).
