American Society of Hematology

Fig. 3.

$Fig. 3. (A) Endothelial cell CD44 partitions into caveolin containing low-density membrane fractions. ECV304 cells grown in 10-cm Petri dishes were extracted in TKM-0.5% TX-100 (upper panel) or 60 mmol/L OTG (lower panel) at 4°C for 30 minutes. The lysates were subjected to equilibrium sedimentation, and the fractions were tested for the distribution of CD44, CD59, or caveolin as described in Fig 1. Caveolin was analyzed under reducing conditions. (B) CD44 and CD59 do not interact directly in the low-density membrane fractions. Surface biotinylated ECV304 cells were extracted with TKM-0.5% TX-100 and ultracentrifuged in sucrose gradients, and the GPI-rich top fractions 3 through 6 were pooled. One milliliter of the pool was precleared with Pansorbin and immunoprecipitated with protein A/G beads coated with antibodies against CD59 or CD44. SDS-PAGE separated proteins were detected by Western blot using streptavidin-HRP.$

(A) Endothelial cell CD44 partitions into caveolin containing low-density membrane fractions. ECV304 cells grown in 10-cm Petri dishes were extracted in TKM-0.5% TX-100 (upper panel) or 60 mmol/L OTG (lower panel) at 4°C for 30 minutes. The lysates were subjected to equilibrium sedimentation, and the fractions were tested for the distribution of CD44, CD59, or caveolin as described in Fig 1. Caveolin was analyzed under reducing conditions. (B) CD44 and CD59 do not interact directly in the low-density membrane fractions. Surface biotinylated ECV304 cells were extracted with TKM-0.5% TX-100 and ultracentrifuged in sucrose gradients, and the GPI-rich top fractions 3 through 6 were pooled. One milliliter of the pool was precleared with Pansorbin and immunoprecipitated with protein A/G beads coated with antibodies against CD59 or CD44. SDS-PAGE separated proteins were detected by Western blot using streptavidin-HRP.

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