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Fig. 1. Localisation of the human CNTF-R and endogenous caveolin-1 in stably transfected MDCK cells. (A,B) MDCK cells stably transfected with the human CNTF-R were grown on collagen-coated coverslips for 5 days. Indirect immunofluorescence staining was performed using specific antibodies against the CNTF-R and caveolin-1 followed by Cy3-conjugated (CNTF-R) and FITC-conjugated (caveolin-1) secondary antibodies. Microscopy was performed using a Zeiss Axiovert 100 M confocal laser scanning microscope equipped with LSM 5 Pascal (Jena, Germany). (A) The xy-scan shows the localisation of the human CNTF-R (red) and caveolin-1 (green) in serial sections of 0.4 µm from the basal to the apical pole. (B) The xz-scan for CNTF-R and caveolin-1. (C) Parental MDCK (lanes 1 and 4) and stably transfected MDCK-CNTF-R cells (lanes 2+3 and 5+6) were grown on Transwell filters for 5 days. Sulfo-NHS-biotin was employed to selectively label the apical or the basolateral surface. The cells were extracted with lysis buffer and the supernatants were immunoprecipitated with a monoclonal CNTF-R-specific antibody. Immunoprecipitates were analysed by SDS-PAGE and western blot. A molecular mass marker containing albumin (66 kDa) was used for comparison. The biotinylated proteins were detected using HRP-conjugated streptavidin and visualised by ECL+plus. For MDCK-CNTF-R cells two independent determinations are shown.
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