Fig. 3. Depletion of PH_CRAC-GFP from the cytosol after uniform stimulation with very low cAMP concentrations. Cells expressing PH_CRAC-GFP were stimulated in a perfusion chamber with the indicated concentration of cAMP. (A) Images before (top) and 14 seconds after (bottom) stimulation with 0.3 nM cAMP, showing one cell with PH_CRAC-GFP patches (left) and two cells with a more uniform cytosolic distribution of PH_CRAC-GFP (right). Scale bar: 10 µm. (B) Quantification of the decrease of fluorescence intensity of the cytosol after cAMP stimulation in cells with or without a PH_CRAC-GFP patch. The results show the means ± s.d. of 10 to 22 cells per cAMP concentration from two independent experiments. (C) Frequency distribution of the number of cells with different amounts of depletion of fluorescence intensity in the cytosol after stimulation with 0.1, 0.3 or 1 nM cAMP for cells with or without a PH_CRAC-GFP patch.