TRPM8 activation leads to the internalization of Rap1. (Ai) Representative confocal time-lapse analysis of HMECs transfected with 1 μg GFP-Rap1 (green), 2 μg TRPM8 (blue), and 1 μg ER-DsRed to label the ER (red). On the right, the merged images are shown. (Aii and Aiii) Enlargements of the PM interface region displaying the internalization effect of TRMP8 activation on Rap1. The PM is shown by the dotted line in gray. On the right, the surface plot analysis is shown. (Bi and Biii) Plot profile of Rap1, ER, and TRPM8 signals in HMECs under control conditions and after 10 min of treatment with 10 µM icilin. On the left is a photomicrograph of the cell in with a line indicating the cross section used for the plot profile. (Bii and Biv) Plots showing the means from a representative experiment of the distances measured between Rap1 and ER (red), between Rap1 and TRPM8 (blue), and between Rap1 and the PM (black) in HMECs under control conditions and after 10 min of treatment with 10 µM icilin. The distances were calculated as means of at least three different plot profiles for at least three regions of interest (cross sections) within each EC.