Figure 5.

ErbB2 is a raft-associated receptor. (A) Western blot of ErbB2 in SKBR3 cells after detergent centrifugation by using different detergents (Tx-100, Brij58, and Brij98). S, supernatant; P, pellet. Below the blot is shown the densitometry measurements of three to five experiments for each detergent (mean ± SD). Black bars represent supernatant and gray bars pellet. (B) Western blot of ErbB2 in SKBR3 cells after cholesterol depletion with 8 mM mβCD for 30 min at 37°C with subsequent Brij98 detergent centrifugation. The densitometry measurements (of three experiments shown as mean ± SD) below show that ErbB2 is moved from the insoluble fraction (the pellet) to the soluble fraction (the supernatant) upon cyclodextrin treatment. (C) Western blots of ErbB2 with or without pretreatment of the cells with 8 mM mβCD for 30 min at 37°C followed by extraction with Brij98 at 37°C and subsequent sucrose density gradient centrifugation. It is seen that cholesterol depletion moves the low buoyancy fraction of ErbB2 down in the gradient, characteristic of raft-associated proteins.