Simultaneous cytosolic and ER Ca²⁺ monitoring in WT or mutant cells. (A) Cytosolic and ER Ca²⁺ signals were monitored with G-GECO1.1 (top) and R-CEPIA1er (bottom), respectively. Representative fluorescence signals in WT (left), R2473S (middle), and K4750Q (right) cells. The numbers 1 and 2 near the left bottom trace mark luminal Ca²⁺ thresholds 1 and 2. (B) Bar graph comparing luminal Ca²⁺ threshold 1 and 2 in WT, R2473S, and K4750Q cells. n = 155 (WT), 122 (R2473S), and 107 (K4750Q). ***, P < 0.001 versus WT threshold 1. ^###, P < 0.001 versus WT threshold 2. Values shown are mean ± SEM.