Figure 1.

Specific interaction of VISTA with galectin-9. VISTA was captured on the enzyme-linked immunosorbent assays (ELISA) plate using goat anti-VISTA antibody. It was then exposed to medium used to culture phorbol 12-myristate 13-acetate (PMA)-pre-treated (24 h with 100 nM PMA) THP-1 acute myeloid leukemia (AML) cells, secreting galectin-9, or MCF-7 breast cancer cells which do not secrete this protein. Bound proteins were then extracted and subjected to Western blot analysis of galectin-9 (A). Alternatively, the format was subjected to ELISA-based detection of galectin-9 (B). VISTA protein expression was measured using Western blot analysis in primary human NK cells and Jurkat T cells (C). Binding of human recombinant VISTA (used as a fusion protein with human Ig-Fc) and galectin-9 was analyzed using synchrotron radiation circular dichroism (SRCD) spectroscopy. The spectrum of human Ig-Fc was subtracted from the fusion protein VISTA-Fc (D). Binding affinity of galectin-9 to VISTA was analyzed using SRCD titration using a fixed concentration of VISTA-Fc and increasing concentrations of galectin-9 (E). The same experiment was performed using human Ig-Fc instead of VISTA-Fc (F). Images are from one experiment representative of five which gave similar results. Data are shown as mean values ± SEM of five individual experiments. SRCD experiments were performed six times each and average results are presented; *p < 0.05 vs control.