Supplemental Figure

This article contains the following supporting material:

• Supplemental Figure.pdf - Effect of cholesterol and sphingolipid depletion on DRM-association of caveolin 1 in cav 1/PrPC FRT cells. A) FRT cells co-expressing PrPC and caveolin 1 were grown to confluence on 100 mm dishes and were lysed for 20 min in cold TNE/TX-100 buffer and then run through a discontinuous 5-40% sucrose gradient. One ml fractions (12 fractions in total) were collected from the top to bottom of the tube after centrifugation to equilibrium, and proteins were TCA precipitated from all fractions, loaded on 12% gels and PrPC and caveolin 1 were revealed by western blotting with specific antibodies and ECL. B) Sucrose gradients of FRT cells co-expressing PrPC and caveolin 1 were performed after treatment with mevinolin and b-cyclodextrin (Mev/bCD) or Fumonisin B1 (FB1) as previously described (Taraboulos et al., 1995; Keller and Simons, 1998; Naslavsky et al., 1999). Proteins were TCA precipitated from all fractions, loaded on 12% gels and caveolin 1 was revealed by western blotting with a specific antibody and ECL. Note that both PrPC and cav 1 float to similar DRM fractions (A). However, the flotation of cav 1 (which is present mainly in the Golgi and at the cell surface) is affected both by cholesterol and sphingolipid depletion (B) like the mature PrP form and not like the immature ER precursor.
  

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