1. The LuTHy procedure
The LuTHy procedure Schematic representation of the workflow of the LuTHy method. Expression vectors encoding NL and PA‐mCit‐tagged hybrid proteins are cloned and co‐transfected into mammalian cells. Binary interactions are detected with a double readout; first, in vivo with in‐cell bioluminescence resonance energy transfer (BRET) and second, ex vivo with a luminescence‐based co‐precipitation (LuC).Schematic representation of control proteins and fusion proteins of interest for investigating the interaction between PA‐mCit‐BAD and NL‐BCL2L1 in proof‐of‐principle LuTHy experiments.Calculated BRET ratios for the indicated protein pairs. The positive control protein PA‐mCit‐NL and the interacting fusion proteins NL‐BCL2L1 and PA‐mCit‐BAD show high BRET ratios.Calculated LuC ratios for the indicated protein pairs. The positive control proteins PA‐NL and PA‐mCit‐NL and the interacting proteins NL‐BCL2L1 and PA‐mCit‐BAD show high LuC ratios.Data information: Data are representative of more than three independent experiments. All values are mean ± s.d. Significance was calculated by one‐way ANOVA followed by Dunnett's multiple comparisons post hoc test; ***P < 0.001.
Philipp Trepte et al. Mol Syst Biol 2018;14:e8071
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