The exocytosis rate was determined as described previously (13). Briefly, cells were preincubated at 37°C with saturating concentration of anti-HA antibody to label YFP-HA-Orai1 and fixed after various incubation times. Initially, Orai1 at the PM was labeled, and an increase in total cell-associated anti-HA antibodies was observed over time as more Orai1 trafficked to the PM and interacted with anti-HA antibodies. Cells were then fixed, permeabilized, and stained with Cy5-labeled anti-mouse antibody. Therefore, the time course of the Cy5/YFP ratio reflects the exocytosis of YFP-HA-Orai1. Data were fit to a monoexponential growth curve as discussed previously to determine the exocytosis rate (17).

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