BRET ratios for 4KO 293T cells co-expressing TCR-Nluc and TCR-GFP combined with 4KO 293T cells expressing BTN2A1 or BTN2A1-BTN3A1-BTN3A2 complex overnight following 4 h treatment with or without HMBPP (10 M) before read. Readings were started immediately after furimazine addition. Since the HMBPP did not cause a significant difference in the signal of the 4KO 293T cells expressing only 2-E2A-9-Nluc and 2-E2A-9-GFP, the HMBPP-treated reporter cells were chosen as the control group and used as the baseline for comparison. The other groups are presented as ratios relative to this baseline. The experiments were conducted in three independent replicates. Data are means SEM of at least three independent experiments, each performed in triplicate. One-way ANOVA analysis was used to calculate significance in bar graphs as indicated. Differences were considered as statistically significant whenp< 0.05 (*p 0.05,**p 0.01,***p 0.001,****p 0.0001, and ns for no significance). Please also seeFigure S6.
