N methyl glucamine

Chemical reagents were sourced as follows: fluo-4 AM (Thermo Fisher); fluo-3 pentapotassium salt (Biotium). HEPES, CHAPS, potassium gluconate, N-methylglucamine, ATP, and DTT (Sigma Aldrich); cOmplete™ EDTA-free protease inhibitor cocktail (Roche); ryanodine (Enzo Life Sciences); . FLI-06 (cyclohexyl 2,7,7-trimethyl-4-(4-nitrophenyl)-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate, C₂₅H₃₀N₂O₅), tyrphostin AG538 (AG538, α-Cyano-(3,4-dihydroxy)cinnamoyl-(3ʹ,4ʹ-dihydroxyphenyl)ketone, C₁₆HN₁₁O₅) and I-O-Me-Tyrphostin AG538 (I-O-Me-AG538, α-Cyano-(3-methoxy-4-hydroxy-5-iodocinnamoyl)-(3′,4′-dihydroxyphenyl)ketone, C₁₇H₁₂INO₅) were purchased from Cayman Chemical, Sigma Aldrich, and Santa Cruz Biotechnology respectively. Caffeine and IP₃ were purchased from Sigma Aldrich. cADPR, 8-NH₂-cADPR, and NAADP were synthesized in house using previously detailed STAR methods (Jain et al., 2010 (link); Walseth and Lee, 1993 (link)). Drug libraries used for the sea urchin screen were from Sigma (LOPAC^®1280, Library of Pharmacologically Active Compounds, 1280 compounds) and Selleck (GPCR compound library, 254 compounds). FLI-06 analogs were sourced from ChemBridge. A Human Notch1 reporter assay (BPS Bioscience) was used per manufacturer's instructions.

OPTIClear was prepared as described previously [14]. Briefly, a 20% (w/v) N‐methylglucamine (Sigma‐Aldrich #66930), 32% (w/v) Iohexol (Sigma‐Aldrich #D2158) and 25% (v/v) 2,2’‐thiodiethanol (TDE) (Sigma‐Aldrich #88559) solution using distilled water was made with pH adjusted to seven using concentrated hydrochloric acid.
RIMS [2], ScaleS4 [7], PROTOS [11], SeeDB2S [16], OPTIVIEW [17] and CUBICScale2 [18] were all prepared as described previously.