Modified BRET using Nluc allowed reduction in protein expression levels, but did not enable observation of ligand-induced BRET ratio.

A) HEK293FT cells were transfected with 100 ng of TLR1/2, MyD88 and TIRAP constructs (TLR pathway) and 0, 100, 200 or 400 ng of NF-κB luciferase constructs, and treated with 1 μg/ml Pam3CSK4. NF-κB luciferase levels measured after 24 hr; representative experiment of n = 3. B and C) HEK293FT cells were transfected with 200 ng of NF-κB-Fluc constructs and 0, 25, 50 or 100 ng of TLR1/2, MyD88 and TIRAP constructs (TLR pathway), and treated with 1 μg/ml Pam3CSK4. NF-κB reporter Fluc luciferase (B) and TLR-Rluc8 luciferase (C) levels measured after 24 hr; representative experiment of n = 2. HEK293FT cells transfected with D) 50 ng TLR1 + 50 ng TLR2-Nluc + 50 ng MyD88-Venus + 100 ng TIRAP constructs, or E) 5 ng TLR1 + 5 ng TLR2-Nluc + 5 ng MyD88-Venus + 10 ng TIRAP constructs. Cells were stimulated with 1 μg/ml Pam3CSK4 (red) or vehicle control (blue) and BRET measured over time. Data shown are donor-only-subtracted BRET ratio; graphs are representative of three independent experiments. F) HEK293FT cells were transfected with 100 ng TLR1 + 100 ng TLR2 + 10 ng MyD88-Nluc and 10 ng (circle) or 50 ng (triangle) of TIRAP-Venus constructs. Cells were stimulated with 1 μg/ml Pam3CSK4 (red and purple) or vehicle control (blue and green) and BRET measured over time. Data shown are donor-only-subtracted BRET ratio; graphs are representative of two independent experiments. G) HEK293FT cells were transfected with constant (100 ng) TLR1/2 and MyD88-Nluc constructs (20 ng, blue; or 10 ng, green), and increasing (0–600 ng) TIRAP-Venus construct. Saturation curves were fitted using ‘one site—specific binding’ function on Prism software.