Fig 2: Dynamic interaction of SARAF with Orai1 and STIM1.MEG-01 cells were suspended in HBS containing 300 μM CaCl2 and were left untreated or stimulated with TG (1 μM) for 30 or 120 s. The cells were then fixed with 4% ice-cold paraformaldehyde and blocked using blocking solution from the Duolink In Situ Red kit® from Sigma. Samples were incubated with primary antibodies: rabbit anti-SARAF and mouse anti-STIM1 or mouse anti-Orai1 (SAB4200273, Sigma) antibodies. A Duolink assay was subsequently performed according to the manufacturer’s instructions as described in Material and Methods. After performing the PLA procedure, images were taken with an Eclipse TE300 fluorescence microscope. Red spots represent individual dimers. The positive control is shown in the lower right corner. The bars indicate 10 μm. The bar graphs indicate association between SARAF and STIM1 or Orai1 expressed as the mean ± S.E.M. of 100–200 cells. The quantification of PLA signals was performed using ImageJ software. ***p < 0.001.

Fig 3: Analysis of the Orai1 site that interacts with SARAF.NG115-401L cells were transfected with SARAF alone or in combination with either pEYFP-full-length Orai1 (FL-Orai1), the N-terminal deletion mutants (pEYFP-Orai1 ΔN1–38 (Orai1 ΔN1–38), ΔN1–72 (Orai1 ΔN1–72) and ΔN1–89 (Orai1 ΔN1–89) (a), pEYFP-Orai1 C-terminal deletion mutant (Orai1-ΔCtermin, amino acids 1–260 (b,c) or empty vector (Mock), as indicated. After 48 h cells were lysed and the whole cell lysates were immunoprecipitated (IP) with anti-SARAF antibody (a,b) or anti-YFP antibody (c). Immunoprecipitates were subjected to 10% SDS-PAGE and subsequent Western blotting with a specific anti-Orai1 (amino acids 288–301, Sigma) antibody (a), anti-Orai1 (ab177021, Abcam) antibody (b) or anti-SARAF antibody (c). Membranes were reprobed with the antibody used for immunoprecipitation for protein loading control (middle panels). (a,b) Alternatively, the cell lysates were subjected to 10% SDS-PAGE and subsequent Western blotting with anti-Orai1 antibody (specific for amino acids 288–301; panel a, bottom) or with anti-Orai1 (ab177021, Abcam; panel b, bottom). HC, heavy chain of the antibody used for immunoprecipitation. The panels show results from one experiment representative of 3 others. Molecular masses indicated on the right were determined using molecular-mass markers run in the same gel.

Fig 4: SARAF impairs OASF-dependent Orai1 function.NG115-401L cells were transfected with pEYPF-Orai1 and pEYPF-OASF in the absence of presence of SARAF or siSARAF or were mock treated, as indicated. After 48h cells were perfused with a Ca2+-free medium (100 μM EGTA added) followed by reintroduction of external Ca2+ (final concentration 1 mM) to initiate constitutive Ca2+ entry. Data are original traces representative of 42–58 experiments. Values are expressed as described in methods.

Fig 5: SARAF regulates TG-induced SOCE and STIM1-Orai1 interaction.(a,b) MEG-01 cells overexpressing SARAF and mock-treated cells (a) or MEG-01 cells transfected with si SARAF or scramble plasmid (b) were perfused with a Ca2+-free medium (100 µM EGTA added) and then stimulated with TG (1 µM) followed by reintroduction of external Ca2+ (final concentration 300 µM) to initiate Ca2+ entry. Data are original traces representative of 42-58 experiments. Values are expressed as described in methods. The bar graph represents TG-induced Ca2+ entry as mean ± SEM and data are presented as percentage of control. (c) Cells overexpressing SARAF, treated with siSARAF and mock-treated cells were lysed and subjected to Western blotting with anti-SARAF antibody, followed by reprobing with anti-actin antibody for protein loading control. (d) MEG-01 cells overexpressing SARAF or transfected with si SARAF and their respective controls were stimulated with TG (1 µM) in a Ca2+-free medium (100 µM EGTA added) and three min later lysed. Whole cell lysates were immunoprecipitated (IP) with anti-STIM1 antibody and immunoprecipitates were subjected to 10% SDS-PAGE and subsequent Western blotting with a specific anti-Orai1 (aa 288–301 (Sigma)) antibody. Membranes were reprobed with the antibody used for immunoprecipitation for protein loading control. The panels show results from one experiment representative of 6–7 others. Molecular masses indicated on the right were determined using molecular-mass markers run in the same gel. HC, heavy chain of the antibody used for immunoprecipitation. The bar graph represents the quantification of STIM1-Orai1 association in resting and TG-treated cells. Results are recorded as arbitrary optical density units, expressed as mean ± S.E.M. and presented as percentage of control. * and *** represent p < 0.05 and p < 0.001, as compared to their respective controls.