Fig. 5

Enhanced bystander BRET in vitro shows potential NPR-11 internalization. A NPR-11 might undergo agonist-promoted endocytosis in vitro while the membrane marker remains at the plasma mebrane. A genetic fusion of NPR-11 with a cyan fluorescent protein (eCFP) and a mNeonGreen anchored to the plasma membrane via the CAAX motif was expressed heterologously in HEK293 cells. Cells were stimulated with 100 nM TAM-FLP-34-1, subcellular localization was monitored in live cells after 30 min, 60 min, and additionally 30 min and 60 min after agonist wash-out. Cell nuclei were stained with Hoechst33342. B Membrane fluorescence measurement (x-fold of before stimulation) of NPR-11::eCFP (left panel) and CAAX::mNG (right panel) before stimulation, 30 min and 60 min before and after agonist wash-out with cells treated as described in A. While the fluorescence of CAAX::mNG remains stable over time, the fluorescence of NPR-11::eCFP drops significantly after 60 min of stimulation suggesting peptide-mediated internalization. Fluorescence levels recover after agonist wash-out. Shown is the mean ± SEM in n = 3 assays with N ≥ 10 cells per condition. * p ≤ 0.05, one-way ANOVA, Dunnett‘s post test against 0 min. Scale bars = 10 μm