TRPM8 activation promotes GDP-binding with Rap. (A–C) Active Rap1 pull-down assay on HMECs transfected with 1 µg control vector (HMEC-WT; A), 1 µg TRPM8, 1 µg TRPM8^Y905A, or 1 µg Rap1-N17 (C) were treated or not with 10 µM icilin or 40 ng/ml PSA (B) for 10 min. Positive control: GTPγS; negative control: GDP. Lower bands refer to total Rap1. (Aii, Bii, and Cii) quantification of the Rap1 activity quantified as Rap1GTP over total Rap1 normalized to HMEC-WT condition (Aii) or untreated conditions (Bii and Cii). (D) Quantification of adherent EC to FN of HMECs transfected with 0.7, 1.0, 1.6, and 2.5 µg Rap1-N17 or control vector, treated or not with 10 µM icilin. *, P < 0.05. The data show adhesion in the icilin-treatment group normalized to control conditions. (E) Quantification of adhesion to FN of HMECs transfected with 1 µg Rap1-N17 and/or 0.5 or 2 µg TRPM8, treated or not with 10 µM icilin. *, P < 0.05. The data show adhesion in the icilin-treatment group normalized to control conditions. (F and G) Quantification of the mean relative velocity of a virtual cell expressing basal 0.2 µM TRPM8, treated or not with icilin, after the addition of different amounts of Rap1-N17 and/or of exogenous TRPM8. The values shown are means of 100 simulations. Error bars show standard deviations. The data show velocity in the icilin-treatment group normalized to control conditions.