Figure 5.

Regulation of membrane cholesterol by Ly49Q through its sulfatide binding ability. (A) Phase-contrast microscopy view of WEHI3 transfectants after treatment with 5 mM mβCD for 20 min. (B) WEHI3 transfectants were treated with different concentrations of mβCD as indicated for the indicated periods. The frequencies of PI-positive cells were examined by flow cytometry. (C) Schematic view of GST-fusion proteins prepared for analyzing the lipid binding ability of Ly49Q. The amino acids shown in red at the top represent the consensus motifs of CARC and CRAC, and YKK in the CRAC motif was substituted with AAA, as shown at the bottom. (D) The lipid binding ability of GST-fused Ly49Q was examined using Sphingo strips. Positions of lipid species spots are shown on the right. (E) The frequencies of PI-positive WEHI3 transfectant cells expressing the Ly49Q-ΔYKK mutant after treatment with mβCD were examined by flow cytometry. All results shown are representative of at least two separate experiments.