TRPM8 activation promotes internalization of Rap1 WT, but not Rap1-GDP. Live-cell imaging of ECs performed using confocal microscopy. Representatives pictures of ECs cotransfected with ER-DsRed and Rap1-WT-GFP 1 µg (Rap1WT; A) or Rap1-N17-GFP 1 µg (Rap1N17; B) at t = 0 and after 10 min of treatment with 10 µM icilin. Rap1 is labeled in green, ER is labeled in red, and PM is marked in gray. In the surface plot (on the right), it is possible to appreciate the colocalization of Rap1 and ER. We chose to label the ER for experimental simplicity, as we have previously demonstrated (Fig. 5) that TRPM8 is closely related to the ER in terms of localization and dynamics. (C) Histograms showing the means of the distances measured in plot profile between Rap1 and ER (red) and between Rap1 and the PM (gray) in ECs transfected with Rap1-WT and Rap1-N17.