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HEK 293 T cells were seeded into T75 flasks at 90,000 cells/cm2 and incubated at 37 °C and 5% CO2 overnight. The following day cells were transiently transfected for 24 hours. IP1 accumulation was measured using Homogeneous Time-Resolved Fluorescence (HTRF) IP-ONE Tb Kit (Cisbio, Codolet, France) as per manufacturer’s instruction. The assay was perfomed using 10,000 cells/well into white, low volume 384-well plates (Greiner Bio-One) The HTRF signal was read on an Envision imager (Perkin-Elmer) after a 1 hour room temperature incubation.
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