Figure 1. C123S mutant CD28 is expressed on the cell surface but it binds to the ligand B7-1 with a lower affinity than the wild-type.
CD28 constructs were transiently expressed in HEK293T cells as YFP fusion proteins. A) On SDS-PAGE, C123S CD28 shows a single band under both reduced and non-reduced conditions equivalent with the monomeric form, as opposed to the wild-type CD28 that is a dimer under non-reduced conditions. B) Both C123S mutant and wild-type CD28 are expressed equally well on the cell surface. Cells transfected with wild-type (filled histogram) and C123S (solid line) CD28, as well as mock-transfected (dashed line) cells stained with an anti-mouse CD28 monoclonal antibody (37.51). C) Binding assay with B7-1-Ig. D) Binding assay with B7-2-Ig. Detection was performed using anti-Fc-Cy5 secondary staining. Normalized MFI values are plotted after substracting the background MFI of mock-transfected cells.