Plasmids pDS-YFP-HA-Orai1, Orai1-ΔN, and Orai1-ΔC were a gift from R. Lewis (Stanford University, Palo Alto, CA) (8, 33). Plasmid expressing GFP-CCT2 was a gift from J. Grantham (The Wenner-Gren Institute, Stockholm University, Sweden) (49). HA-NFAT1(4-460)–GFP (Addgene plasmid #11107) and human STIM1-CFP (Addgene plasmid #19755) were from A. Rao’s Lab (La Jolla Institute for Allergy and Immunology, La Jolla, CA). To construct pDS-YFP-HA-Orai1(1-266), pDS-YFP-HA-Orai1(1-275), and pDS-YFP-HA-Orai1(1-285), nucleotides at respective positions were substituted to generate premature stop codon using a site-directed mutagenesis kit from Stratagene. All constructs were validated by sequencing. The primers used were as follows: for YFP-HAOrai1-ΔC (1-256), 5′-TCTTCGCCGTCCACTAGTACCGCTCACTGGTT-3′ (forward) and 5′-AACCAGTGAGCGGTACTAGTGGACGGCGAAGAC-3′ (reverse); for YFP-HA-Orai1 (1-266), 5′-GCTCACTGGTTAGCCATAAGACTTAGCGACAGTTCCAG-3′ (forward) and 5′-CTGGAACTGTCGCTAAGTCTTATGGCTAACCAGTGAGC-3′ (reverse); for YFP-HA-Orai1(1-275), 5′-CAGGAGCTCAACGAGTAGGCGGAGTTTGCCCG-3′ (forward) and 5′-CGGGCAAACTCCGCCTACTCGTTGAGCTCCTG-3′ (reverse); for YFP-HA-Orai1 (1-285), 5′-GCTTACAGGACCAGTAGGACCACAGAGGGGA-3′ (forward) and 5′-TCCCCTCTGTGGTCCTACTGGTCCTGTAAGCG-3′ (reverse).

For generation of scrambled 157-167(ICL-S) Orai1 mutant, the QIAGEN LongRange PCR Kit (Qiagen) was used according to the manufacturer’s protocol. Briefly, polymerase chain reaction was done with 20 ng of template pDS-YFP-HA-Orai1 DNA using the primers 5′-PO4-AACGAGAAGCCCCATCGCTCGCTCGTCGAGTCCATGCACCGC-3′ (forward) and 5′-PO4-ATTGTGCACGTTGCTCACCGCCTCGATGTTGGGCAGGATGCAGGTGCT-3′ (reverse) and 35 cycles of the following amplification protocol: 3-min initial activation at 93°C, 35 cycles (15-s denaturation at 93°C, 30-s annealing at 62°C, and 10-min primer extension at 68°C).

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