Calcium Signaling in Engineered Cell Lines

Cyclopiazonic acid (CPA) was from Tocris (Bristol, UK). D-myo-inositol 1,4,5- trisphosphate (IP₃) was from Enzo (Exeter, UK). Synthetic adenophostin A [24 ] was a generous gift from Prof Barry V. L. Potter, University of Oxford. Mag-Fluo-4 AM, Mag-Fluo-4 tetrapotassium salt, rabbit Fluorescein/Oregon Green polyclonal antibody (QAb, catalogue number A-889), water-soluble probenecid, RPMI medium and Dulbecco's modified Eagle’s medium/nutrient mixture F-12 with GlutaMAX (DMEM/F-12 GlutaMAX) were from ThermoFisher Scientific (Paisley, UK). Plasmid encoding the ER-targeted GECI, G-CEPIA1er [25 (link)], was from Addgene #58215. DT40 cells lacking endogenous IP₃Rs were from Dr T. Kurosaki (Kansai Medical University, Japan) [26 (link)]. HEK-293 cells were from Kerafast (Boston, USA). TransIT-LT1 transfection reagent was from Geneflow (Elmhurst, Lichfield, UK). G418 was from Formedium (Norfolk, UK). Half-area 96-well black-walled plates were from Greiner Bio One (Stonehouse, UK). Unless otherwise specified, other reagents, including porcine liver carboxylesterase (EC 3.1.1.1) and foetal bovine serum (FBS), were from Sigma-Aldrich (Gillingham, UK).