Fig 1.
(A) VEGF quantification method of the eBRET2 VEGF biosensor. A conformational change after VEGF binding leads to an increased energy transfer and thus to a higher BRET signal, in dependence of the VEGF concentration. (B) Overlapping Rluc8 emission wavelength and GFP2 excitation wavelength generates GFP2 emission at higher wavelength. (C) Biosensor expression constructs Re01–Re10. CMV = cytomegalovirus Promoter, Rluc8 = Renilla luciferase, R = VEGF receptor, D = IgG domain, GFP2 = green fluorescent protein, pA = polyadenylation signal. VEGF receptor 1 (R1) and 2 (R2) structure. The receptors contain a transmembrane-, an intracellular catalytic domain and 7 extracellular IgG like domains. IgG like domain 2 (D2) and 3 (D3) are responsible for the VEGF binding and were introduced between the Rluc8 and the GFP2 as VEGF binding domain.
Fig 2.
Characterization of eBRET2 biosensor expression.
(A) Fluorescence microscopy of GFP2 expression after biosensor plasmid transfection in HEK293 T cells. (B) Luciferase activity via Luminescence measurement. Rluc8 expression correlates with Luciferase activity. (C) eBRET2 ratios of the biosensor variants normalized with the correction factor (Rluc8 29.8 ± 0.3 mBU). Significance defined as p < 0.001. (D) Western Blot shows full length expression of the biosensors and correction factor (Rluc8 alone). 1. Antibody: anti Rluc8 (1:1000) on the top and anti GAPDH (1:1000) on the bottom. 2. Antibody: anti Rabbit HRP (1:20000). Arrow indicates additional bands that show extra splice variants. (E) Splice prediction with Human Splicing Finder 3.0 software. Circles represent donor sites, squares represent acceptor sites, and strongest candidates are located in the grey area. Arrow marked the strong donor splice site in the Rluc8. (F) Western Blot after deletion of the splice site shows full length expression of the biosensors and correction factor (Rluc8 alone). No splice variants could be detected. The box shows the base sequence of the splice site with one letter code for the corresponding amino acids above. In red marked the change of the thymine base into an adenine base to delete the splice site. The amino acid sequence did not change.
Fig 3.
Characterization of eBRET2 biosensor expression after the deletion of the splice site.
(A) Luciferase activity via Luminescence measurement. Rluc8 expression correlates with Luciferase activity. (B) Western Blot shows full length expression of the biosensors, correction factor (Rluc8 alone) and maximum control Rluc8 GFP2. 1. Antibody: anti Rluc8 (1:1000) on the top and anti GAPDH (1:1000) on the bottom. 2. Antibody: anti Rabbit HRP (1:12000). (C) eBRET2 ratios of the biosensor variants normalized with the correction factor (Rluc8 30.1 ± 0.1 mBU). Significance defined as p < 0.001. (D) Delta eBRET2 ratios after incubation with 10 ng VEGF. Change in ΔBR of the maximum control (Rluc8 GFP2) was set as detection limit, marked with the line.
Fig 4.
VEGF dependent Delta eBRET2 Ratios of the four biosensors Re02 (A), Re06 (B), Re07 (C) and Re09 (D). Biosensor concentrations in cell lysates were adjusted on the basis of RLuc8 expression to 200,000 RLU and 400,000 RLU. Lysates were incubated with individually adjusted VEGF concentrations and Delta eBRET2 Ratios were calculated. Significance defined as p < 0.05.