Human c5a

ELISAs for human C5a (#DY2037; R&D, Minneapolis, MN, USA), mouse C5a (#EK0987; Boster, Pleasanton, CA, USA), mouse IL-6 (#431304; BioLegend Inc., San Diego, CA, USA), mouse matrix metalloproteinase-3 (MMP-3, #MMP300; R&D), and mouse anti-citrullinated protein antibody (ACPA) (#ORG601 Orgentec, Chicago, IL, USA) in serum were performed in accordance with the instructions of the manufacturers. The limits of detection for each analyte were as follows: human C5a, 31.3 pg/mL; mouse C5a, 15.6 pg/mL; mouse IL-6, 7.8 pg/mL; mouse MMP-3, 0.312 ng/mL; and mouse ACPA, 0 U/mL.

Apigenin was purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). Monoclonal antibody against C5aR (sc-271949) was supplied by Santa Cruz Biotechnology (Dallas, TX, U.S.A.). Monoclonal antibodies against human PCAF (3378), STAT3 (9139), and β-actin (3700) were from Cell Signaling Technology (Danvers, MA, U.S.A.). Horseradish peroxidase-conjugated anti-mouse IgG (7076) and anti-rabbit IgG (7074), as well as ECL detection system were purchased from Cell Signaling Technology. PVDF membranes were from Millipore (Billerica, MA, U.S.A.). The pcDNA3.1 vector was from Invitrogen. The incision enzymes of HindIII and BamHI as well as T4 DNA ligase were purchased from TaKaRa (Tokyo, Japan). Human C5a was from R&D Systems (Minneapolis, MN, U.S.A.). Cell counting kit-8 (CCK-8) was purchased from Dojindo Laboratories (Kumamoto, Japan). FuGENE®HD was from Promega (Madison, WI, U.S.A.).