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Fig. 8. Analysis of Triton solubility of GFP-22-Nglyc and GFP-22-MutNglyc. (A)
Detergent lysates prepared from MDCK cells expressing GFP-22-Nglyc (upper part
of panel) or GFP-22-MutNglyc (lower part of panel) were brought to 40% sucrose
and analysed by flotation through a sucrose step gradient, as described in
Materials and Methods. Half of each fraction was analysed by western blotting,
using anti-GFP, anti caveolin I or anti-E cadherin antibodies, as indicated.
(B) Half of fraction 4 and 1/20th of fraction 11 (11*) from the
same gradient of panel (A) were analysed by western blotting with anti-GFP
antibodies. Bracket, arrow and arrowhead in (A) and (B) have the same meaning
as in Fig. 7C. Note enrichment
of mature glycosylated form of GFP-22-Nglyc in fraction 4, which corresponds
to the interface between the 30% and 5% sucrose layers and contains
raft-associated caveolin.
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