Figure 3.

Nanobody specificity validation in mammalian cells. a) Schematic of the M2H assay. DrBphP and nanobody genes were inserted into the bait and prey plasmids, respectively. b) Specificity comparison of LID systems. HEK293T cells were transiently co-transfected with the bait, prey, and GAL4UAS-luciferase reporter plasmids (~0.25 μg each) in a 0.5 mL culture (no biliverdin added). None, the negative control transfected with only the bait and the luciferase reporter plasmids. Cells were maintained under the illumination (654 nm (0.2 mW/cm²) or 775 nm (0.2 mW/cm²)) or in the dark for 24 hours before measuring luciferase levels. Different from DrBphP, RpBphP1 is required to be converted to the light form by a NIR (e.g., 775-nm) light. Data represent mean values of 3 measurements; error bars, standard deviation.