Figure 4. The interaction between the Nt and lipid bilayers.

(A) Liposome co-sedimentation assay. Nt was produced as a fusion protein with glutathione transferase (GST-Nt) in the BL21 strain of Escherichia coli and purified with glutathione-Sepharose 4B beads. GST was used as a control. Diagram showing the liposome sedimentation assay. GST and and GST-Nt were centrifuged before use to sediment insoluble proteins and mixed with liposomes (input). After centrifugation, the liposome-bound (ppt) and liposome-unbound (sup) fractions were recovered. Aliquots of each fraction were separated on an SDS-PAGE and stained with Coomassie brilliant blue. (B) Lipid binding assay using the spot array method. Lipid-spotted membrane strips were incubated with GST or GST-Nt and the lipid-bound proteins were detected using anti-GST antibody. LPA, lysophosphatidic acid; PI, phosphatidylinositol; PI(3)P, phosphatidylinositol-(3)-phosphate; PI(4)P, phosphatidylinositol-(4)-phosphate; PI(5), phosphatidylinositol-(5)-phosphate; PE, phosphatidylethanolamine, PC, phosphatidylcholine; SIP, sphingosine-1-phosphate; PI(3,4)P₂; phosphatidylinositol-(3,4)-bisphosphate; PI(3,5)P₂, phosphatidylinositol-(3,5)-bisphosphate; PI(3,4,5)P₃, phosphatidylinositol-(3,4,5)-trisphosphate; PA, phosphatidic acid; PS, phosphatidylserine.