Fig. 3.

Anti-citrullinated protein antibodies (ACPAs) promote the interaction between CD147 and integrin β1. a After ACPA treatment, phorbol-12-myristate-13-acetate (PMA)-differentiated THP-1 cells were probed with an anti-human CD147 antibody, an fluorescein isothiocyanate (FITC)-conjugated anti-human immunoglobulin G (IgG) secondary antibody, and a Cy3-conjugated anti-mouse IgG secondary antibody. The colocalization of ACPAs and CD147 was evaluated by confocal laser scanning microscopy. Scale bars = 10 μM. b After IgG treatment, PMA-differentiated THP-1 cells were probed with an anti-human CD147 antibody, an FITC-conjugated anti-human IgG secondary antibody, and a Cy3-conjugated anti-mouse IgG secondary antibody. The colocalization of control IgG and CD147 was evaluated by confocal laser scanning microscopy. Scale bars = 10 μM. c Biophysical analysis of the CD147/ACPA interaction using surface plasmon resonance. The orange, green, peach, blue, violet purple, and red lines represent different concentrations of CD147 (0, 0.5, 1, 2, 4, and 8 μM, respectively) in the fluid phase. d Lysates of PMA-differentiated THP-1 cells were subjected to immunoprecipitation with a coupling gel prebound to ACPA IgG. Mouse IgG was used as the negative control. CD147 in the precipitate was detected by western blotting (upper image). ACPA IgG and control IgG were subjected to immunoprecipitation with an anti-CD147 antibody. IgG in the precipitate was detected by western blotting (lower image). e–g After preincubation with ACPA IgG or control IgG, lysates of PMA-differentiated THP-1 cells with CD147 or ITGB1 knockdown or GRGDS treatment were subjected to immunoprecipitation with a coupling gel prebound to an anti-CD147 antibody (e), an anti-ITGB1 antibody (f), or an anti-human IgG antibody (g). ITGB1 and CD147 in the precipitate were detected by western blotting