(A) Western blot and matching autoradiogram of RPE-1 cells expressing ORAI1-YFP plus the indicated PAT isoform, labelled with 3H-palmitic acid and immunoprecipitated with anti-GFP. Graph bar (right) shows densitometry analysis of the tritiated bands relative to GFP in RPE1 (Blue, N = 2) and HeLa cells (yellow, N = 1). (B) Functional effect of PAT3, 7, and 20 expression. Representative western blot of HeLa cells expressing Myc-tagged PAT isoforms (left), averaged SOCE responses (middle), and peak amplitude (right). Data are mean ± SEM of 49–74 cells from three independent experiments. (C) Averaged SOCE responses of WT (left) or C143A (middle) S1/O1 cells expressing these PAT isoforms and their peak amplitude (right). Data are mean ± SEM of 31–129 cells from five independent experiments. (D) Lipid partitioning of ORAI1 in giant plasma membrane vesicles from HEK-293 cells transiently transfected with PiP2-Cherry and WT or C143 ORAI1-YFP together with PAT20 or PCDNA3. Left: representative fluorescence images of vesicles from cells expressing PAT20 and WT or C143 ORAI1-YFP (green) stained with cholera toxin subunit B (Magenta) as raft marker and PiP2-Cherry (Red) as non-raft marker (scale bar = 2.5 µm). Right: Graph bar representing the % of ORAI1 preference for raft domains. Data are mean ± SEM of 13 (WT empty), 12 (C143A empty), 23 (WT+ PAT20), and 16 (C143A + PAT20) vesicles from four independent experiments. (A–C) One-way ANOVA Dunnett’s multiple comparisons test. (D) One-tailed unpaired Student’s t-test.