Fig 1: Orai1α modulates Mn2+ influx through TRPC1 and TRPC1 plasma membrane expression. a–d Representative responses to 2 µM TG in HeLa cells co-transfected with STIM1, Orai1α, Orai1β and TRPC1 or the corresponding dominant-negative Orai1 mutants, as described. Cells were super-fused with HBSS containing 0.5 mM Mn2+ and 1 mM Ca2+ and stimulated with 2 µM TG (indicated by arrow). Fura-2 fluorescence was measured at an excitation wavelength of 360 nm, the isoemissive wavelength. Representative traces were chosen to represent the datasets. e Quantification of the rate of decay of fura-2 fluorescence under the different experimental conditions (from left to right, n = 28, 32, 22 and 40; n values correspond to individual cells). Scatter plots are represented as mean ± SEM and were statistically analyzed using Kruskal–Wallis test with multiple comparisons (Dunn’s test). **p < 0.01 and ***p < 0.001 as compared to HeLa cells expressing STIM1, Orai1α, Orai1β and TRPC1. $p < 0.05, statistical significance among the responses observed in cells expressing dnOrai1α or dnOrai1β mutants. f HeLa cells were co-transfected with STIM1-CFP, Orai1α (or dnOrai1α mutant, as indicated), Orai1β (or dnOrai1β mutant, as indicated) and TRPC1 (or with shTRPC1, as indicated). Forty-eight hours later, cells were mixed with biotinylation buffer containing EZ-Link sulfo-NHS-LC-biotin, and cell surface proteins were labeled by biotinylation, as described in “Material and methods”. Labeled proteins were pulled down with streptavidin-coated agarose beads. The pellet (containing the plasma membrane fraction) was analyzed by SDS-PAGE and Western blotting using anti-TRPC1 or anti-PMCA antibody, as indicated. Molecular masses indicated on the right were determined using molecular-mass markers run in the same gel. These results are representative of seven separate experiments. g Quantification of TRPC1 plasma membrane expression under the different experimental conditions normalized to the PMCA expression. Scatter plots are represented as mean ± SEM and were statistically analyzed using Kruskal–Wallis test with multiple comparisons (Dunn’s test). **p < 0.01 and ***p < 0.001 as compared to HeLa cells expressing STIM1, Orai1α, Orai1β and TRPC1. $p < 0.05, statistical significance among the TRPC1 surface expression in cells expressing dnOrai1α or dnOrai1β mutants
Fig 2: Interaction of Orai1α and Orai1β with TRPC1 and STIM1. HeLa cells were suspended in HBS containing 1 mM Ca2+ and then stimulated for 1 min with 2 µM TG (TG) or the vehicle (C) and lysed. Whole-cell lysates were immuno-precipitated with anti-TRPC1 (a) or anti-STIM1 antibody (b). Immuno-precipitates were treated with PNGaseF and then subjected to 10% SDS-PAGE and Western blotting with the anti-Orai1 antibody, as described in Material and Methods. Membranes were re-probed with the antibody used for immunoprecipitation for protein loading control. Molecular masses indicated on the right were determined using molecular-mass markers run in the same gel. Blots are representative of four to seven separate experiments. c, d Scatter plots, representing the quantification of the TRPC1-Orai1α/β or STIM1-Orai1α/β interaction, are presented as mean ± SEM and were statistically analyzed using the Mann–Whitney U test (*p < 0.05 and **p < 0.01, as compared to the corresponding control (C)). e HeLa cells were co-transfected with STIM1-CFP, TRPC1 and either Orai1α-GFP (lanes 1 and 2) or Orai1β-GFP (lanes 3 and 4). Cells were suspended in HBS containing 1 mM Ca2+ and then stimulated with 3 µM histamine (lanes 2 and 4) or the vehicle (lanes 1 and 3) for 5 min, and the interaction between Orai1 variants and TRPC1 was assessed by APEX2 proximity labeling assay, as described in Material and Methods. The biotinylated and non-biotinylated fractions were subjected to 10% SDS-PAGE and Western blotting with the anti-TRPC1 antibody. The non-biotinylated fraction was also probed with the anti-Orai1antibody for protein loading control. Molecular masses indicated on the right were determined using molecular-mass markers run in the same gel. Blots are representative of three separate experiments. f Scatter plots, representing the quantification of the TRPC1-Orai1α or TRPC1-Orai1β interaction in resting (C; control) and histamine (H)-treated cells, are presented as mean ± SEM and were statistically analyzed using the Mann–Whitney U test (*p < 0.05, as compared to the corresponding control). HC heavy chain of the IgG used for immunoprecipitation
Fig 3: Effect of Orai1 inhibition and expression of the STIM1 (K684,685E) mutant on Mn2+ influx in HeLa cells. a Representative responses to 2 µM TG in HeLa cells co-transfected with STIM1, Orai1 and TRPC1, in the absence or presence of synta66 (10 µM), or co-transfected with STIM1, Orai1 and shTRPC1, as described. Cells were super-fused with HBSS containing 0.5 mM Mn2+ and 1 mM Ca2+ and stimulated with 2 µM TG (indicated by arrow). Fura-2 fluorescence was measured at an excitation wavelength of 360 nm, the isoemissive wavelength. Representative traces were chosen to represent the datasets. b Quantification of the rate of decay of fura-2 fluorescence under the different experimental conditions (from left to right, n = 35, 61 and 47; n values correspond to individual cells). Scatter plots are represented as mean ± SEM and were statistically analyzed using Kruskal–Wallis test with multiple comparisons (Dunn’s test). ***p < 0.001 as compared to HeLa cells expressing STIM1, Orai1 and TRPC1. c Representative responses to TG in HeLa cells co-transfected with STIM1 or the STIM1(K684,685E) mutant, Orai1 and TRPC1, as indicated. Cells were super-fused with HBSS containing 0.5 mM Mn2+ and 1 mM Ca2+ and stimulated with 2 µM TG (indicated by arrow). Fura-2 fluorescence was measured at an excitation wavelength of 360 nm, the isoemissive wavelength. Representative traces were chosen to represent the datasets. d Quantification of the rate of decay of fura-2 fluorescence under the different experimental conditions (from left to right, n = 55 and 54; n values correspond to individual cells). Scatter plots are represented as mean ± SEM and were statistically analyzed using Mann–Whitney U test
Supplier Page from MilliporeSigma for Anti-ORAI1 (AB1) antibody produced in rabbit