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. Author manuscript; available in PMC: 2011 Feb 16.
Published in final edited form as: J Biol Chem. 2006 Aug 30;281(45):34601–34609. doi: 10.1074/jbc.M601530200

FIGURE 2. Association of CD44 with low buoyant density lipid raft membrane fractions.

FIGURE 2

Panel A represents a typical Western blotting profile for CD44 expressed by five different selected clones of Flp-293 cells stably transfected with CD44wt. The clones are labeled as C1 through C5, whereas E depicts endogenous levels of CD44 expressed by Flp-293 cells. COS-7 transfectants (B) and Flp-293 cells (D) expressing CD44wt were extracted in the presence of 1% Triton X-100 on ice and subjected to ultracentrifugation on step gradients of iodixanol. Equal volume fractions were collected and analyzed by Western blotting for CD44. The 0–20% iodixanol interface (predominately the 20% fraction) represents the low density, lipid raft fraction. In some instances, cells were cholesterol-depleted by pretreatment with 20 mm Mβ-CD at 37 °C for 50 min for COS-7 cells (C) or 25 min for Flp-293 cells (E) prior to ultracentrifugation and Western blotting. The same fractions from the Flp-293 stable transfectants shown in D and E were also assayed for the lipid raft marker flotillin-1 using the anti-flotillin-1 antibody, before (F) and after (G) cholesterol depletion. CD44 was shown to associate with a low buoyant density lipid raft fraction in both COS-7 and Flp-293 cells (verified by the migration of flotillin-1), and this association was dependent on the presence of cholesterol. CD44 was detected using either an anti-CD44 monoclonal antibody, BU-52 (B and C) or the epitope-tag anti-V5 monoclonal antibody (D and E).