American Society of Hematology

Figure 1.

$Figure 1. Properties of lipid rafts in HEK293 cells. (A) Tissue factor does not stably associate with lipid rafts. Wild-type HEK293 cells (10 million) were lysed in 0.1 mL MES-buffered saline (pH 6.5) containing 1% Triton X-100, adjusted to 40% sucrose, and placed beneath a step gradient consisting of 1.0 mL 30% sucrose and 0.4 mL 5% sucrose. Following centrifugation, gradient fractions were subjected to SDS-PAGE and Western blot using anti-TF (top blot) and anti-DAF (bottom blot) antibodies. Buoyant, Triton-insoluble material is indicated by DAF distribution. (B) Complete disruption of lipid rafts requires treatment with 10 mM MBCD for 60 minutes. Sixty million HEK293 cells expressing GPI-anchored and hemagglutinin (HA)-tagged TFPI were treated with cell buffer (CB) for 60 minutes (top blot), 10 mM MBCD for 30 minutes (middle blot), or 10 mM MBCD for 60 minutes (bottom blot) before addition of 1 mL MES-buffered saline containing 1% Triton X-100. Lysates were adjusted to 40% sucrose and placed beneath a step gradient consisting of 5 mL 30% sucrose and 3 mL 5% sucrose. Following centrifugation, gradient fractions were subjected to SDS-PAGE and Western blot using anti-HA antibody to detect epitope-tagged TFPI.$

Properties of lipid rafts in HEK293 cells. (A) Tissue factor does not stably associate with lipid rafts. Wild-type HEK293 cells (10 million) were lysed in 0.1 mL MES-buffered saline (pH 6.5) containing 1% Triton X-100, adjusted to 40% sucrose, and placed beneath a step gradient consisting of 1.0 mL 30% sucrose and 0.4 mL 5% sucrose. Following centrifugation, gradient fractions were subjected to SDS-PAGE and Western blot using anti-TF (top blot) and anti-DAF (bottom blot) antibodies. Buoyant, Triton-insoluble material is indicated by DAF distribution. (B) Complete disruption of lipid rafts requires treatment with 10 mM MBCD for 60 minutes. Sixty million HEK293 cells expressing GPI-anchored and hemagglutinin (HA)-tagged TFPI were treated with cell buffer (CB) for 60 minutes (top blot), 10 mM MBCD for 30 minutes (middle blot), or 10 mM MBCD for 60 minutes (bottom blot) before addition of 1 mL MES-buffered saline containing 1% Triton X-100. Lysates were adjusted to 40% sucrose and placed beneath a step gradient consisting of 5 mL 30% sucrose and 3 mL 5% sucrose. Following centrifugation, gradient fractions were subjected to SDS-PAGE and Western blot using anti-HA antibody to detect epitope-tagged TFPI.

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