Fig. 1: Plasma membrane binding of PTH^TMR.

A Principle of TIRF experiments. The measurements were performed in 3 different regions of interest (ROI); the bottom membrane, the top membrane, and the cover glass. Created in BioRender. Pacheco Romero, J. (2025) https://BioRender.com/u84v021. B Fluorescence time courses (left) and representative images (right) of CLTA^mNG cells without the expression of the receptor (mock cells) or cells expressing the PTH₁R^mNG. PTH^TMR or TMR fluorophore was added at 30 sec, indicated by the horizontal red bar at the bottom. The mNG channel (in gray) was used to ensure the TIRF focus plane. Experiments are representative of n = 6/3 (PTHR), n = 5/4 (mock) and n = 5/2 cells/independent days of experiment. Scale bar, 5 µm. C Diffusion coefficient comparison between PTH^TMR molecules detected on cover glass with no cells, and HEK293 cells with or without expression of PTH₁R^mNG. The dotted line represents the threshold under which trajectories are excluded from our analysis. Grand mean ± 95% confidence intervals (C.I.) of n = 10/179 (PTH₁R^NG), n = 23/829 (cover glass), and n = 7/3631 (mock) with cells/average trajectories per cell. ****P < 0.0001 by one-way ANOVA with a Tukey’s multiple comparison.