Figure 3

Activation of RAC1 triggered the translocation of ORAI1 to CTTN-containing cell periphery. Panel A: Cells growing onto collagen-coated coverslips in FBS-containing DMEM were transfected for the expression of mCherry-CTTN and EGFP-RAC1 (wt) or EGFP-RAC1^Q61L (active RAC1). At 36 h after transfection, cells were fixed and visualized under wide-field epifluorescence microscopy. Cherry and GFP channels were recorded sequentially using independent filter blocks. The two images were merged, and the fluorescence of both channels was measured over the arrow depicted in the image. Fluorescence values, plotted on right panels, were quantified with the NIS-Elements AR software. Images are representative of a minimum of 32 cells per condition from 3 independent experiments. Bar = 10 μm. Panel B: Cells stably expressing Flag-RAC1 (wild-type), Flag-RAC1^G12V, or Flag-RAC1^T17N were transfected for the expression of mCherry-CTTN and ORAI1-GFP. As in panel A, cells were analyzed under epifluorescence microscopy, and Cherry and GFP channels were recorded. Images are representative of >26 cells per condition from 3 independent experiments. Bar = 10 μm.