Fig. 1.

A: MβCD inhibition of PI4KIIα. A buoyant membrane fraction enriched for PI4KIIα activity and TGN markers was prepared by equilibrium density gradient ultracentrifugation of postnuclear supernatants from COS-7 cells. Membranes were sterol-depleted with MβCD (10 mM) and [³²P]PI4P production determined in the presence of added PI (0–100 μM). B: Desmosterol and enantiomeric-cholesterol can support PI4KIIα-catalyzed PI phosphorylation. Equal volume aliquots of a TGN membrane fraction were sterol-depleted with MβCD and [³²P]PI4P production measured in the presence of either desmosterol or enantiomeric cholesterol added in complex with MβCD. ^*P < 0.05 compared with MβCD-pretreated samples. The final concentration of MβCD in these assays was equivalent to the IC₅₀ concentration for inhibition of PI4P synthesis. MβCD, methyl-β-cyclodextrin; PI, phosphatidylinositol; PI4KII, type II phosphatidylinositol 4-kinase; PI4P, phosphatidylinositol 4-phosphate; TGN, trans-Golgi network.