Figure 9

BRET analysis of B7.1, B7-H1 and Ad5FB4 interactions in situ. (A, B), Interactions between B7.1 and B7-H1 molecules. Homodimeric and heterodimeric interactions were monitored in transfected HeLa cells using BRET donor saturation assays. Both B7.1 and B7-H1 were fused to Rluc8 to act as energy donors, alternatively; B7.1 (black circle), B7-H1 (open circle), and IR (open square) were fused to YPet to act as energy acceptors. Cells were cotransfected with plasmids expressing B7.1-Rluc8 or B7-H1-Rluc8 at constant dose, and with different doses of the other YPet-fused interactors. Results, expressed as mean milliBRET values (mBRET), were plotted versus the values of the YPet-to-Rluc8 ratio. (C, D), Effect of penton-HeLa cell interaction on B7.1-B7-H1 heterodimers. B7.1-RLuc8 and B7-H1-YPet were coexpressed in HeLa cells and the BRET donor saturation curves were determined in the absence (basal BRET; open circle) or presence of increasing doses of penton protein, at 33 (light grey circle), 66 (dark grey circle), and 132 ng/μL (black circle), respectively. (C), Ad5FB4 penton; (D), Ad5 penton. Results, expressed as mBRET, were plotted versus the values of the YPet-to-Rluc8 ratio. Each point of the curves represented the average of six independent experiments (m ± SEM).