Fig. 1: Establishing a direct BRET assay to capture the dynamics in the WNT-3A-induced complex formation between FZD5-Nluc and LRP5/6-Venus. | Nature Communications

Fig. 1: Establishing a direct BRET assay to capture the dynamics in the WNT-3A-induced complex formation between FZD5-Nluc and LRP5/6-Venus.

From: WNT-induced association of Frizzled and LRP6 is not sufficient for the initiation of WNT/β-catenin signaling

Fig. 1

A Architecture of LRP-Venus and FZD-Nluc constructs. SP, signal peptide; FLAG, FLAG-tag; E1-E4, extracellular YWTD β-propeller/EGF domain repeats 1-4; LDL-A, low-density lipoprotein receptor type A repeats; TM, transmembrane domain; ICD, intracellular domain; CRD, cysteine-rich domain; 7TM, seven-transmembrane domain; C-term., receptor C-terminal domain. B Schematic depiction of the assay principle. Parts of this figure were created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDrivs 4.0 International license. Created in BioRender. Voss, J. https://BioRender.com/lfyvjb8. C, D. WNT-3A stimulation (1000 ng/ml) kinetics of HEK293 (C) or HEK293T ΔLRP5/6 (D) cells transfected with FZD5-Nluc and LRP5/6-Venus (yellow and blue circles, respectively; note: y-axes are not scaling equal in all figure panels). E Stimulation of HEK293 cells transfected with FZD5-Nluc and LRP5- or LRP6-Venus with 1 nM of WNT surrogate. F WNT-3A stimulation (1000 ng/ml) of HEK293 cells transfected with FZD5-Nluc and chimeric LRP5/6-Venus variants. G Architecture of LRP6 and LRP6-5A-Venus constructs. PPP(S/T)P/PPPAP, phosphorylation motifs. H. WNT-3A stimulation kinetics of HEK293A cells transfected with FZD5-Nluc and LRP6-/LRP6-5A-Venus (blue and light blue circles, respectively). Data are presented as mean ± standard error of the mean (SEM) of five (C), four (D), or three (E, F, H) individual experiments, each performed in triplicate.

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