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500 500 500 500 400 400 400 400 300 300 300 300 200 200 200 200 100 100 100 100 0 0 0 0 0 0 0 0 200 200 200 200 400 400 400 400 600 600 600 600 800 800 800 800 500 400 300 200 100 0 a b 20 0 Ca 20 Ca Peak Ca2+ TG GFP- Orai1 WT 400 [Ca2+]i (in nM) GFP+ 300 [Ca2+]i (in nM) 200 100 0 Ca 20 Ca 20 20 0 Ca 20 Ca 0 WT E106A resting E106D E190A E106Q TG TG La3+ (2 mM) Orai1 E190A 2-APB (2 mM) [Ca2+]i (in nM) Influx Rate 14 12 10 0 200 400 600 800 1000 8 Influx rate (nM/s) 6 20 0 Ca 20 Ca 20 0 Ca 20 Ca 4 2 TG TG 2-APB (2 mM) 0 Orai1 E106A WT E106A E106D E106Q E190A [Ca2+]i (in nM) 20 0 Ca 20 Ca 20 0 Ca 20 Ca 800 800 TG TG La3+ (2 mM) Orai1 E106D 600 600 [Ca2+]i (in nM) 400 400 200 200 0 0 200 400 600 800 0 0 200 400 600 800 20 0 Ca 20 Ca 20 0 Ca 20 Ca 800 TG 800 TG Orai1 E106Q La3+ (2 mM) 600 600 [Ca2+]i (in nM) 400 400 200 200 0 0 0 200 400 800 600 0 200 400 600 800 Time (s) Suppl Figure 4