Figure 3

Identification of FLRT2 as a novel autoantigen of AECAs. (A) Unsorted, C9, and C18 were stained with isotype control or anti-FLRT2 antibody, followed by secondary antibody, and analyzed with flow cytometry (left). Western blotting of proteins from unsorted, C9, and C18 was performed, and they were stained with anti-FLRT2 antibody followed by secondary antibody (right). Arrows indicate the bands of FLRT2. Both of the two bands are FLRT2 because some of FLRT2 proteins were glycosylated. (B) Expression vector, empty-IRES-GFP, or FLRT2-IRES-GFP was transfected into HEK293T cells, and these cells were stained with 0.5 mg/ml of control IgG or E10-19 IgG, followed by secondary antibody, and analyzed with flow cytometry. Binding activities of IgG to cell-surface FLRT2 were analyzed in histograms (right) by gating for the GFP-positive transfected population (left). (C) HUVECs were stained with isotype control or anti-FLRT2 antibody followed by secondary antibody, and analyzed with flow cytometry (left). Western blotting of proteins from HUVECs was performed, and they were stained with anti-FLRT2 antibody followed by secondary antibody (right). The arrows indicate the bands of FLRT2. (D) HAECs, HRGECs, and HMVEC-Ls were stained with isotype control or anti-FLRT2 antibody followed by secondary antibody, and analyzed with flow cytometry. (E) HUVECs were stained with isotype control, anti-FLRT1 antibody, or anti-FLRT3 antibody followed by secondary antibody, and analyzed with flow cytometry. (F) Expression vector, FLRT1-IRES-GFP (left), or FLRT3-IRES-GFP (right) was transfected into HEK293T cells, and these cells were stained with 0.5 mg/ml of control IgG or E10-19 IgG, followed by secondary antibody, and analyzed with flow cytometry.