FIGURE 4.

Ack1 is required for translocation of TRAIL receptors to lipid rafts. A, lipid raft-containing proteins were analyzed by Western blotting of sucrose gradient fractions of MCF10A cells transfected with non-targeting control (siCtrl) or Ack1 (siAck1) siRNA and subjected to TRAIL or mock treatment. Note that lipid rafts containing caveolin-1 were enriched in low-density fractions 1–3. B, Western blot confirming efficient Ack1 knockdown in the cell lysates used in A. C and D, subcellular fractionation by sequential detergent extraction of MCF10A cells transfected with non-targeting or Ack1 siRNA followed by treatment with TRAIL for the indicated time periods. D, quantification of TRAIL-R1 levels in the soluble membrane versus insoluble cytoskeleton/raft fractions as described for C by densitometry analysis of Western blots (n = 3). Note the Ack1-dependent translocation of TRAIL-R1 to the cytoskeleton/raft fraction following TRAIL treatment. E, quantification of the effect of the lipid raft-disrupting compound methyl β-cyclodextrin (MbCD) on TRAIL-induced cell death by densitometry analysis of Western blots against cleaved caspase-8 (n = 3). F, Western blot showing the effect of pretreating cells with the raft-disrupting drug nystatin on TRAIL-induced Ack1 and cleaved (Cl) caspase-8 (representative of three independent experiments). Note that nystatin prevented TRAIL-induced accumulation of Ack1. Error bars indicate S.E. p values were obtained with Student's t test.