Fig. 4

Enhanced bystander BRET reveals information on receptor trafficking and internalization of NPR-11 in vivo. A Bystander BRET was determined between Nluc fused to the C terminus of NPR-11 (NPR-11::Nluc, donor) and an mNeonGreen fused to the C-terminal part of let-60 (CAAX::mNG, acceptor) expressed in the same individual. B Donor and acceptor are generating a bystander BRET signal that is significantly higher than the donor-only (baseline) BRET ratio. Experiments were set-up as in Fig. 3. Luminescence and fluorescence values are given in Table S1. Shown is the mean ± SEM in n ≥ 5 assays with 30 worms per well as triplicates; * p ≤ 0.05 two-sided t-test. C Enhanced bystander BRET (measured over time) of worms treated either with BSA or 5 μM FLP-34-1, baseline-corrected (value - baseline) for every single time point and pruned rows (mean of every 10 min value with the following value starting with t = 2.5 min). While the control stays stable around the baseline, the netBRET of FLP-34-1 drops after 42.5 min, opening a significant window till 62.5 min (grey area), suggesting a separation of donor and acceptor. Shown is the mean ± SEM in n = 3 assays with 30 worms per well as triplicates; * p ≤ 0.05 two-way ANOVA, Bonferroni‘s posthoc test. Schematic images were created with BioRender.com