Endogenous TRPM8 inhibits Rap1 activity. (Ai) Live-cell imaging by confocal microscopy of ECs expressing GFP-RBD_RalGDS probe in control (siCNTRL) and siTRPM8-treated ECs (siTRPM8). Representative images showing RBD_RalGDS in siCNTRL- and siTRPM8-treated ECs at t = 0 and after 2 or 10 min of treatment with 10 µM icilin; the bottom parts of the figures represent enlargements of the inset (white lines) for each time point. (Aii and Aiii) Bar plot representing quantification of GFP-RBD membrane recruitment calculated as cytosol translocation as described in the method section. *, P < 0.05 (Wilcoxon–Mann-Whitney test). (Bi) Active Rap1 pull-down assay on HMEC or HUVEC silenced for TRPM8 (siTRPM8) or not (siCNTRL), in the presence or absence of icilin. (Bii) Quantification of the Rap1 activity normalized to HMEC siCNTRL or HUVEC siCNTRL condition. Data are expressed as mean ± SEM. *, P < 0.05 (Wilcoxon–Mann-Whitney test).