Figure 1. ANO8 increases interaction of STIM1‐Orai1 and STIM1‐STIM1 at the ER/PM junctions to control Ca²⁺ influx.

1. HEK cells transfected with STIM1‐CFP, Orai1‐mCherry, and with (red) or without (black) untagged ANO8 were used to measure CRAC current with pipette solution (cytoplasmic buffer) containing 3 mM EGTA. The columns show the averaged effect of ANO8 on current density and on the slope of slow Ca²⁺‐dependent inactivation (SCDI). The results are mean ± SEM, and differences were analyzed by unpaired t‐test.
2. Fura2‐loaded HEK cells transfected with YFP (control, black) or ANO8‐YFP (red) were used to measure store‐mediated Ca²⁺ influx. Stores were depleted by treatment with the SERCA inhibitor CPA, and Ca²⁺ influx was measured by Ca²⁺ add‐back. The results are mean ± SEM, and differences were analyzed by unpaired t‐test.
3. FRET efficiency was measured with HEK cells transfected with STIM1‐CFP, ANO8‐YFP, and with (red) and without (black) Orai1‐HA before and after store depletion. Here and all other FRET measurements, representative images of the FRET signal under each condition are shown next to the traces. The averages are given in (D).
4. Summary of FRET efficiency measurements at the indicated conditions. The results are mean ± SEM, and differences were analyzed by unpaired t‐test.
5. FRET efficiency was measured with HEK cells transfected with STIM1‐CFP, STIM1‐YFP (blue), and co‐transfected with untagged ANO8 (green) or treated with siANO8 (red) before and after store depletion.
6. Resting (R) or store‐depleted (S) HEK cells transfected with STIM1‐YFP and myc‐STIM1 and with or without ANO8 or treated with siANO8, were used to immunoprecipitate (IP) STIM1‐YFP and blot (B) for myc‐STIM1. The inputs (In) are with anti‐myc, anti‐YFP, or anti‐ANO8.
7. Confocal and bright‐field images of basal (upper) and store‐depleted cells (lower) of HEK cells transfected with ANO8‐YFP alone.
8. TIRF images (upper) and TIRF‐Z scan (lower) of the same resting and store‐depleted HEK cells transfected with ANO8‐YFP alone.
9. Confocal images of resting cells transfected with ANO8‐YFP, STIM1‐CFP, and Orai1‐mCherry.
10. Confocal images of store‐depleted cells transfected with ANO8‐YFP, STIM1‐CFP, and Orai1‐mCherry.
11. TIRF‐Z scan of resting (upper) and store‐depleted (lower) cells transfected with ANO8‐YFP and STIM1‐mCherry demonstrating the relationship between STIM1 and ANO8 puncta at the TIRF field. Cell surface is on top.

Data information: The first number in parenthesis indicates the number of similar experiments performed, and the second number is the number of cells analyzed. All results are given as mean ± SEM of the indicated number of experiments or cells analyzed. The experiments in (G–K) represent at least four separate experiments with similar results.Source data are available online for this figure.