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Figure S3. FCCS analyses in MDCK cells and the diffusion coefficients of AQP2 and actin. (A) FCCS analysis in cells expressing DsRed-GFP chimera. (B) FCCS analysis in cells coexpressing DsRed and GFP. The crosshair in each left image indicates the measurement position. Images for GFP (blue) and DsRed (red) were overlayed. Bars, 5 µm. Autocorrelation curves are shown in each right panel: green line corresponds to GFP signal, red line corresponds to DsRed signal, black line is the cross-correlation curve. The dashed lines are measured curves and the solid lines are their fitted curves. Insets show the fluorescence intensities of GFP and DsRed in the two respective channels during an FCCS measurement. CR, count rates. (C and D) FCCS analysis in the subapical region (C) and the apical membrane (D) in the cell expressing DsRed-AQP2 and GFP-actin after forskolin stimulation. C and D show autocorrelation curves at the position indicated in the images shown in Figure 2 (A and D), respectively. Blue lines correspond to GFP-actin signal, red lines correspond to DsRed-AQP2 signal, and black lines are cross-correlation curves. (E and F) Summary of diffusion coefficients of DsRed-AQP2 (E) and GFP-actin (F). C, control; FK, forskolin treatment; H89, H89-pretreated cells expressing DsRed-AQP2 and GFP-actin; S256A, the cells expressing DsRed-S256A-AQP2 and GFP-actin; subapical, measurements in the subapical region; apical, measurements on the apical membrane. Data represent the mean and SE from at least five independent experiments. The diffusion coefficient of AQP2 on the apical membrane was significantly decreased compared with the value in the subapical region (**, P < 0.01). Forskolin stimulation decreased the diffusion coefficient in both the subapical region and the apical membrane; however, only the effect in the subapical region was significant (subapical: *, P < 0.05; apical: P = 0.12).