Figure 4.

Nephrocystin and inversin localize to primary cilia in renal tubular epithelial cells. (a) Colocalization of nephrocystin and β-tubulin-4 in primary cilia of MDCK cells. Two examples of cilia are shown in the upper and lower panels. Wild-type MDCK cells (clone II) were grown on coverslips at 100% confluence and cultivated for 7 d before the experiment to allow full polarization and cilia formation. Localization of nephrocystin was determined by immunofluorescence using nephrocystin-specific antibody with confocal images captured at the level of the apical membrane. Cells were costained with rabbit antibody to nephrocystin (left panels) and mouse antibody to β-tubulin-4 (middle panels) followed by the respective secondary antibodies. (b) Specific localization of nephrocystin in primary cilia was confirmed by the use of blocking recombinant nephrocystin protein. (c) Inversin localizes to primary cilia in MDCK cells. Localization of endogenous inversin was determined by immunofluorescence using inversin-specific antibody with confocal images captured at the level of the apical membrane. Cells were costained with mouse antibody to β-tubulin-4 and rabbit antibody to inversin followed by the respective secondary antibodies (lower panel). In additional stainings, the antibody to β-tubulin-4 was omitted to reduce potential spectral overlap between the inversin and β-tubulin-4 signals (upper panel). (d) Partial colocalization of nephrocystin and inversin in primary cilia. Localization of nephrocystin was determined by immunofluorescence using nephrocystin-specific antibody with confocal images captured at the level of the apical membrane. Cells were costained with goat antibody to inversin (left panel) and rabbit antibody to nephrocystin (middle panel) followed by the respective secondary antibodies. Partial colocalization is shown in the right panel.