(A) Retinal tissues and retinal pigment epithelial (RPE) cells were isolated from WT and CDCP1-KO mice, and cell lysates were prepared. CDCP1 was detected by Western blotting with a polyclonal Ab. Here, CDCP1 was detectable in the retina and purified RPE cells from WT but not CDCP1-KO mice. Actin blots were used as loading controls. (B) Immunofluorescent staining with the anti-CDCP1 mAb clone 9A2 showed selective CDCP1 expression (red) on RPE cells in mouse and human retinas. DAPI (green) was used for nuclear staining. Scale bars: 10 μm (low-magnification images); 1 μm (high-magnification images). (C) CDCP1 expression was detected on human aRPE-19 and hTERT-RPE cells by flow cytometry with both our mAb clone 9A2 and a commercial anti-human CDCP1 mAb, clone CUB1. Gray shades indicate isotype controls; black shades indicate anti-CDCP1 IgGs. (D) CDCP1 expression on RPE cells was upregulated by treatment with IFN-γ but not IL-17 or TNF-α. RPE-19 cells were cultured with 1,000 U each of IFN-γ, IL-17, or TNF-α for 48 hours, after which CDCP1 expression was detected using flow cytometry. (E) CDCP1 expression on RPE was upregulated in vivo in EAU, as detected by immunofluorescent staining of the RPE flat mounts from WT naive and EAU mice, and using the CDCP1-KO mouse tissue as the negative control. 3D reconstruction and the orthogonal view analyses together with ezrin staining confirmed the surface expression pattern of CDCP1 on the RPE cells. CDCP1 levels (MFI) were quantitated from 4 random areas using Fiji software. Scale bars: 50 μm. Each experiment was repeated at least 3 times.