XB-IMG-123485
Xenbase Image ID: 123485
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Figure 2. The C-terminus of Stim (C-Stim) constitutively activates Orai without forming punctaea, b Time course (upper) and I-V curves (lower) comparing CRAC current in representative cells co-transfected with Stim + GFP-Orai (a) or with C-Stim + GFP-Orai (b). Application of 5 nM Gd3+ reversibly blocked most of the current in cells transfected with either Stim + GFP-Orai or C-Stim + GFP-Orai. c, Subcellular localization of GFP-Orai together with Stim-V5His or C-Stim-V5His in resting (StimV5His and C-StimV5His) or store-depleted (2 μM TG for 15 min, Stim-V5His only) co-transfected S2 cells. Puncta formation was only observed upon store-depletion in cells expressing StimV5His and GFP-Orai. The panels underneath each picture display fluorescence intensity profiles (F) for Orai and Stim obtained from the same regions of interest, tracing the perimeter of each cell clockwise from top. To quantify the extent to which GFP-Orai was inhomogeneously distributed we calculated the ratio of fluorescence variance/mean fluorescence from profiles like those illustrated. Mean ratio values for GFP-Orai (±SEM; n=6 cells for each condition) were: StimV5His, -TG, 29.03±2.9; StimV5His, +TG, 62.9±4.5 (p = 0.00002); C-Stim-V5His, -TG, 22.4±3.7 (not significantly different from StimV5His). d, BMH cross-linking in intact S2 cells transfected with HA-Orai only, C-Stim-V5His only or co-transfected. Numbers represent inferred state of oligomerization: 1, monomer; 2, dimer; 3, trimer; 4, tetramer; *, higher order aggregates. The cross-linking pattern of Orai showed a clear tetrameric population when co-expressed with C-Stim, but not in the absence of C-Stim. The cross-linking profile of C-Stim was not affected by the presence of Orai, and revealed a majority of dimers and trimers. Image published in: Penna A et al. (2008) Image downloaded from an Open Access article in PubMed Central. Image reproduced on Xenbase with permission of the publisher and the copyright holder. Larger Image Printer Friendly View |