Stim1

The Orai1–YFP plasmid has been previously described [23 (link), 36 (link)]. STIM1-CFP, YFP–CAD, and CFP–CAD were kind gifts of Dr R. Lewis (Stanford University, USA). Unlabeled STIM1 used for the electrophysiology was obtained from Origene Technologies (Rockville, MD). Cysteine mutations were introduced at Orai1 residues L273 or L276 via site-directed mutagenesis using the QuickChange Mutagenesis Kit (Stratagene) and confirmed via DNA sequencing. FRET to assess binding between Orai1 and STIM1 (or CAD) was performed with YFP-labelled Orai1 constructs and STIM1-CFP or CFP-CAD.
For imaging studies, STIM1–CFP (or CFP–CAD), 100 ng, and the indicated Orai1-YFP, 100 ng, constructs were co-transfected using Lipofectamine 2000 (Life Technologies) and imaged 24 h later. Due to poor transfection efficiency of CFP-CAD, YFP-CAD was co-expressed with Orai1-YFP for Western experiments to ensure maximal CAD occupancy of the Orai1 channels. For electrophysiology experiments, unlabelled STIM1 (300 ng) was co-transfected with Orai-YFP (100 ng) constructs using Transpass D2 (New England Biolabs) and cells were patch-clamped 24 hrs later.

Isolated CD8⁺ T cells were incubated in the presence of 2 mM of Ca²⁺, fixed with paraformaldehyde (Sigma-Aldrich), permeabilized using Triton X-100 (Sigma-Aldrich) and stained with anti-PMCA (Abcam), anti-TMEM20 (SantaCruz Biotechnology) or anti-STIM1 (Cell Signaling Technology) antibody. Then the samples were stained with anti-rabbit IgG-Alex Fluor^® 488, anti-rabbit-IgG-Alex Fluor^® 546 or IgG-Alex Fluor^® 647 (Life Technologies, Gaithersburg, MD) for PMCA, STIM1 or TMEM20, respectively. The images were obtained using LSM 510 META Laser Scanning Microscopes (Carl Zeiss).