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Fig. 3. FRAP analysis of NHE3-EGFP in the three regions in OK cells. Fluorescence photobleaching was performed and recovery analyzed in the three regions shown in Fig. 2 – intracellular JN, apical surface above-JN and non-JN of NHE3-EGFP in OK cells. Images show prebleaching (A), immediately post bleaching (B) and after recovery (450 seconds after bleaching) (C) at the level of the apical plasma membrane. The prebleaching intensity (Fi), intensity just after bleaching (F0) and final intensity after full recovery (F{infty}) are shown in D. FRAP data were collected every 9 seconds up to 50 images. The mobile fraction (Mf) and effective diffusion coefficient (Deff) of NHE3-EGFP in the three pools in OK cells were obtained by fitting the photobleaching curve according to Materials and Methods (see E and F). n=12 cells studied for Cyto-JN, n=15 for above-JN and n=21 for non-JN. Data were from three separate experiments. P values for Mf were in comparison to cyto-JN (unpaired t-test).





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