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Fig. 4. Addition of cholesterol affects the oligomeric state and the polarity of GFP-PrP but does not influence the behaviour of GFP-FR. MDCK cells stably expressing GFP-FR or GFP-PrP were loaded with 2 mM cholesterol (+chol) or not (control), and oligomerisation state and distribution of GFP-FR and GFP-PrP at the plasma membrane were assessed. (A) Cells were lysed as decribed for Fig. 3B and ran through 20-40% glycerol gradients. Fractions of 300 µl were collected from the top (fraction 2) to the bottom (fraction 15) of the gradients. Proteins were precipitated with TCA and detected by western blotting using anti-GFP antibody. The position on the gradients of the molecular mass markers is indicated. (B,C) Plasma membrane localisation was determined by analysing (B) the natural fluorescence of GFP or by (C) an immunofluorescence assay performed under non-permeabilising conditions by adding anti-GFP antibody to the apical side of cells that had been grown on filter for 4 days. Serial confocal sections were collected from the top to the bottom of the cells. Mean fluorescence intensities at the apical and basolateral domains were measured and are expressed as percentages of total fluorescence. Bars, 15 µm.
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