Fig. 2. Anti-uPAR antibody competes with uPA for uPAR binding and inhibits uPAR-dependent signalings in GC cells.
(A) Binding affinity (KD) of the anti-uPAR mAb to human uPAR assessed by SPR. (B and C) IF (B) and immunoelectron microscopy (C) analysis of anti-uPAR binding to uPAR-expressing HEK-293T cells. DAPI, 4′,6-diamidino-2-phenylindole. (D and E) Detection of different forms of uPAR by the anti-uPAR mAb shown by FCM (D) and Western blotting (E). MFI, mean fluorescence intensity. (F) Competitive binding assay showing the ability of anti-uPAR to inhibit uPA binding to uPAR, as shown by FCM. (G) Western blotting of phosphorylated ERK (p-ERK) and total ERK (T-ERK) in SNU-216 cells pretreated with anti-uPAR or CTRL mAb after pro-uPA stimulation. (H to K) Growth curves (H and I), transwell invasion assay (J), and cell adhesion assay (K) of AGS and SNU-216 cells treated with anti-uPAR or CTRL mAb. Data are expressed as means ± SEM (*P < 0.05, **P < 0.01, and ***P < 0.001).