The impact of deleting GRK6 on Ca²⁺release in MEG-01 cells. (A) MEG-01 cells were loaded with fluo-4 AM, and the cells were stimulated with thrombin (n = 6), PAR1-AP (n = 5), PAR4-AP (n = 8), U46619 (n = 5), or collagen (n = 3), and changes of Ca²⁺ were recorded. (Ai) The representative trace of Ca²⁺ response in response to agonists. (Aii) The bar graph summarizes 3 to 8 experiments, respectively (mean ± SEM). (Bi) PAR1 surface expression in WT vs GRK6^−/− MEG-01 cells in unstimulated (left) or in response to thrombin stimulation (right), and (Bii) the percentage decrease in PAR1 surface expression in WT and GRK6^−/− MEG-01 cells after stimulation (N = 3). An anti-PAR1 antibody (ATAP2), which recognizes cleaved as well as intact PAR1 receptors, was used to detect PAR1 surface expression by flow cytometry.