Figure 4.

Detection of close proximity between H₄R and TSPAN4 at the surface of HEK293T cells using bimolecular fluorescence complementation. (A,B) In-frame fusion of the nonfluorescent complementary mVenus fragments V1 and V2 to the intracellular C-terminal tail of H₄R and N-terminal tail of TSPAN4 allows functional reconstitution of mVenus that is driven by the close proximity between H₄R and TSPAN4. (C,E) HEK293T cells transiently co-transfected with H₄R-V1 and V2-TSPAN4. Two days post-transfection, the cells were observed under microscope upon stimulation with vehicle (C) or 10 µM histamine (E). Interaction between H₄R-V1 and V2-TSPAN4 reconstitutes mVenus (green), nuclei are stained with 4’6-diamidino-2-phenylindole (blue). (D) Image of cells two days after transient transfection with mVenus-TSPAN4. Representative images (C–E) are shown from three independent experiment performed in triplicate. (F) Fluorescence intensity of the reconstituted mVenus upon stimulation with vehicle or 10 µM histamine was measured by plate reader at 497-15 excitation and 540-20 emission. Dotted line represents the background fluorescence in mock transfected cells. Data are displayed as mean ± SD from 3 independent experiments performed in triplicate. Statistical difference was analyzed using Student’s t-test. NS = no significant difference.