Orai1 endocytosis rate was measured as the amount of Orai1 internalized as a function of time as described previously (13). Briefly, cells were incubated with saturating concentration of anti-HA antibody, and at various times, cells were fixed and stained with Cy5-labeled anti-mouse secondary antibody to label the Orai1 pool at the PM. Cells were fixed again, permeabilized, and stained with Cy3-labeled anti-mouse IgG to reveal anti-HA that has been internalized. The fluorescence intensities of the Cy3 and Cy5 were measured using quantitative fluorescence microscopy. Unlabeled cells were used for a background correction. The (Cy3/YFP)Internal/(Cy5/YFP)Surface ratio was plotted over time and fitted with a linear regression function.

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