Additional file 1: Figure S1. Protein topology of hTIGIT and hPVR, along with the structure of TIGIT/PVR complex. (a, b) hTIGIT and hPVR contained ectodomain (blue), transmembrane domain (red) and intracellular domain (purple). The glycosylation sites of the extracellular domain were marked with a yellow ball. (c, d) The residue sequences of hTIGIT and hPVR, where the secondary structure α-helix and β-sheet were noted by cyan and yellow shadow respectively, and the residues forming the disulfide bond were represented by red. (e) Model of hTIGIT complexed with hPVR, which was retrieved from heterotetrameric hTIGIT/hPVR complex (PDB ID code 3UDW). The secondary structure and disulfide bond were shown in the same way as above. Figure S2. Stable cell lines of hPVR mutants. (a) CHOK1 cells overexpressing WT and mutant hPVR were stained with anti-human PVR APC. For each histogram, the filled blue histogram with blue line is the hPVR specific antibody and the histogram with red lines is the isotype control. (b) Lysates of CHOK1, CHOK1-hPVR, and CHOK1-mutants were used for western blotting. The blot was developed by chemiluminescence. Figure S3. Relationship between protein expression and binding affinity. Protein expression were normalized versus wild type hPVR cells. Graphs showed the correlation between protein expression and binding affinity When hTIGIT-Fc at different concentrations. The Pearson correlation coefficient and P were shown. Figure S4. Binding affinity of PVR mutants fused with EGFP to hTIGIT-His. The membrane protein of PVR mutants fused with EGFP was used to detect the binding affinity with hTIGIT-His. The concentration of TIGIT-His was serially diluted by two-fold with 0.000153 μM from 25 μM. The KD values of PVR mutants with hTIGIT-His were shown. Graphs were representative of three independent experiments..