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Fig. 4. Immunolocalization of AQP2 in cells pretreated with the ERM peptide. (A) Cells were either left untreated or stimulated with forskolin in the presence or absence of either ERM peptide or the control peptide. Fluorescence was visualized by epifluorescence microscopy and the xz reconstructions were obtained by deconvolution using Autodeblur software (shown in the insets). Forskolin stimulation caused AQP2 translocation from an intracellular pool to the apical membrane. A similar effect was observed on preincubation with the ERM peptide, while control peptide had no effect on AQP2 cellular localization. (B) Ratios of cell membrane/intracellular membrane fluorescence signals. Signal immunofluorescence intensities were detected from deconvoluted cells (Autodeblur software). The intracellular and cell membrane immunofluorescence signal intensities were calculated by using Metamorph software and normalized to the background signal intensities (n=15 for the control, n=13 for forskolin, n=13 for peptide control, n=13 for peptide control in the presence of forskolin, n=15 for ERM peptide, n=15 for ERM peptide in the presence of forskolin). Ratios greater than 1 indicate a cell membrane localization of AQP2. (#P<0.001 with respect to control). Values are expressed as means ± s.e.
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