To measure surface Orai1, cells were washed once with PBS and immediately fixed using freshly prepared 4% (w/v) paraformaldehyde for 10 min at room temperature. In parallel, intracellular Orai1 was detected using cells fixed followed by permeabilization for 10 min in PBS containing 0.1% (w/v) Triton X-100. All subsequent antibody incubations were performed in PBS containing 5% (w/v) FBS. Cells were stained with saturating concentrations of monoclonal anti-HA antibodies (Covance) at 1:300 for 45 min at 37°C, followed by incubation in a 1:400 dilution of Cy5-conjugated goat anti-mouse IgG (Invitrogen) for 30 min at 37°C. The Cy5/YFP ratio was determined by quantitative fluorescence microscopy. In parallel, nonpermeabilized cells were fixed and stained. Then, Cy5/YFP was measured in both cell populations. The background signal of each of the YFP and Cy5 channels was set using two controls: YFP-HA-Orai1–transfected cells incubated only with Cy5 secondary antibody and cells that did not express the YFP-HA-Orai1 construct. The fraction of Orai1 at the PM was determined as the (Cy5/YFP in nonpermeabilized cells)/(Cy5/YFP in permeabilized cells) ratio.

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