CAX regulates cytosolic Ca²⁺ signaling. (a–f) Effect of CAX overexpression on agonist-evoked Ca²⁺ signals. (a) Cytosolic Ca²⁺ levels of SHSY5Y cells loaded with the Ca²⁺ indicator Fura-2 and stimulated with 100 µM carbachol. Cells were from cultures transiently transfected with mRFP-CAX or LAMP1-mRFP, and data were segregated according to whether they were mRFP positive (transfected) or not (untransfected). (b) Pooled data (from seven transfections) quantifying the area under the curve (integral) and the peak response. (c) Effect of mRFP-CAX on carbachol-evoked Ca²⁺ signals in the presence of GPN. Cultures were treated with DMSO or 200 µM GPN for 30 min before stimulation. (d) Pooled data from four transfections. (e) Cytosolic Ca²⁺ responses of cells stimulated with 10 µM carbachol from cultures transiently transfected with LAMP1-mRFP, CAX-mRFP, CAXΔN-mRFP, or mRFP-CAX^E420A. Insets are expanded views of the initial responses presented as line plots without error bars (traces from cells expressing LAMP1 and the CAX mutants overlap). (f) Pooled data from six transfections. (g and h) Effect of CAX knockdown on agonist-evoked Ca²⁺ signals. (g) Cytosolic Ca²⁺ levels of neural crest cells expressing the Ca²⁺ indicator R-GECO1 and stimulated with 1 mM carbachol. Explants were from control embryos and embryos injected with 10 ng AMO1. (h) Pooled data from five knockdowns quantifying the proportion of cells that responded to agonist. Error bars represent SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.