human cfpstim1  (Addgene inc)

Journal: Science signaling

Article Title: ORAI1, stromal interaction molecules 1/2, and ryanodine receptor type 1 shape sub-second Ca 2+ microdomains upon T cell activation

doi: 10.1126/scisignal.aat0358

Figure Lengend Snippet: (A) Ca2+ microdomains in a non-stimulated wt T cell (top), Orai1−/− T cell (middle) and Ryr1−/− T cell (bottom). Arrow heads indicate Ca2+ microdomains directly at the PM. (B) Characteristics of Ca2+ microdomains in primary murine wt (n=69), Orai1−/− (n=28), Stim1−/− (n=24), Stim2−/− (n=39) and Stim1-/−2−/− (n=46) in a 5s time period. Comparison of the number of signals per cell and frame (data represent mean ± SEM). Statistically significant differences are marked by asterisks (* p<0.05, ** p<0.01, *** p<0.001, Kruskal-Wallis Test). (C, D) Kinetic analysis of the 5s time period. Statistical analysis was performed with wt against all other genotypes (as indicated) with Mann-Whitney-U-Test across all time points (**p<0.005, ***p<0.001).

Article Snippet: Both plasmids STIM1 CFP (Addgene plasmid # 19755) and Orai1 YFP (Addgene plasmid # 19756) were gifts from Murali Prakriya (Northwestern University, Chicago) and Anjana Rao (La Jolla Institute, San Diego) ( 38 ).

Techniques: MANN-WHITNEY

Journal: Science signaling

Article Title: ORAI1, stromal interaction molecules 1/2, and ryanodine receptor type 1 shape sub-second Ca 2+ microdomains upon T cell activation

doi: 10.1126/scisignal.aat0358

Figure Lengend Snippet: (A-D) Immunoprecipitation experiments in Jurkat T cells (A, B) and primary murine wt T cells; shown are single representative experiments from at least 3 experiments in Jurkat T cells and primary T cells. (C, D). (A) Detection of STIM1 and ORAI1 in membranes of Jurkat T cells. (B) Representative pull down of fragmented membranes obtained from non-stimulated Jurkat T cells using beads coupled with either anti-ORAI1 (top) or anti-STIM1 (bottom) (-Tg). (C) Detection of STIM1 and ORAI1 in whole cell lysates from primary murine wt T cells. (D) Pull down of fragmented membranes obtained from non-stimulated primary wt T cells using beads coupled with anti-STIM1 (bottom) (-Tg). As positive controls for interaction of ORAI1 and STIM1 in both Jurkat and primary murine T cells, cells were stimulated with thapsigargin (Tg) for 10 min (+Tg). (E) Representative Jurkat T cells double transfected with STIM1-CFP and ORAI1-YFP. Basal STIM CFP, ORAI1 YFP and FRET signal in non-stimulated cells (top) and after 10 min stimulation by thapsigargin (bottom) of double transfected Jurkat T cells. Length scale bar corresponds to 5 μm. (F) Statistical analysis of FRET at the plasma membrane after correction for spectral crosstalk (bleedthrough) from non-stimulated (basal n=20) and thapsigargin stimulated Jurkat cells (n=18). Background corresponds to unspecific FRET within the cytosol of both groups. Data represent mean ± SEM. Statistically significant differences are marked by asterisks (* p<0.05, Kruskal-Wallis Test). G to H: Co-localization of STIM1 (green) and ORAI1 (red) in a representative primary wt T cell, showing single optical channels, merge of both optical channels and co-localization (white). Length scale bar corresponds to 2 μm. (H) Magnification of a high (1) and a low (2) co-localization area of the ROIs depicted in (G); scale bar depicts 100 nm. (I) STIM1-ORAI1 plot profiles of the arrow shown in H1. (J) Statistical colocalization analysis of STIM1-ORAI1 (n=17). Data represent mean ± SEM. Statistically significant differences are marked by asterisks (**** p<0.0001, unpaired t test).

Article Snippet: Both plasmids STIM1 CFP (Addgene plasmid # 19755) and Orai1 YFP (Addgene plasmid # 19756) were gifts from Murali Prakriya (Northwestern University, Chicago) and Anjana Rao (La Jolla Institute, San Diego) ( 38 ).

Techniques: Immunoprecipitation, Transfection

Journal: Science signaling

Article Title: ORAI1, stromal interaction molecules 1/2, and ryanodine receptor type 1 shape sub-second Ca 2+ microdomains upon T cell activation

doi: 10.1126/scisignal.aat0358

Figure Lengend Snippet: (A) Initial Ca2+ microdomains in a wt T cell (top) and a Orai1−/− T cell (bottom) upon anti-CD3/anti-CD28 stimulation (bead contact indicated schematically) and a magnified region near the bead contact site. (B, C) Respective Ca2+ tracings of four ROIs (3×3 pixel) shown in (A top; wt, A bottom Orai1−/−). Dashed line indicates time point of bead contact. (D) Characteristics of initial Ca2+ microdomains in primary murine wt (n=69), Orai1−/− (n=28), Stim1−/− (n=24), Stim2−/− (n=39) and Stim1−/−/Stim2−/− (n=46) in the first 15 s post bead contact. Comparison of the number of signals per cell and frame and the Ca2+ amplitude (data represent mean ± SEM). Statistically significant differences are marked by asterisks (* p<0.05, ** p<0.01, *** p<0.001, Kruskal-Wallis Test). (E) Primary wt, Orai1−/−, Stim1−/−, Stim2−/− and Stim1−/−/Stim2−/− T cells werestimulated by anti-CD3/anti-CD28 coated beads. Subcellular layers at the PM (as indicated) were analyzed in the first second before and post bead contact. Statistical analysis was performed with wt against all other genotypes (as indicated) with Mann-Whitney-U-Test across all time points after activation (*p<0.05, **p<0.005, ***p<0.001). (F) Ca2+ microdomain numbers were compared either before stimulation (−5s to 0s), or in 5s intervals post stimulation as indicated. Data represent mean ± SEM. Statistically significant differences between the number of signals per cell per frame are marked by asterisks (* p < 0.05, ** p<0.01, *** p<0.001, Kruskal-Wallis Test).

Article Snippet: Both plasmids STIM1 CFP (Addgene plasmid # 19755) and Orai1 YFP (Addgene plasmid # 19756) were gifts from Murali Prakriya (Northwestern University, Chicago) and Anjana Rao (La Jolla Institute, San Diego) ( 38 ).

Techniques: MANN-WHITNEY, Activation Assay