LpRalF and RpRalF capping domains associate with membranes by different mechanisms.

<p><b>A</b>) LpRalF and RpRalF capping domains are associated with the cell membrane fraction. HEK293 cells were transfected with YFP-LpRalF_192–374, YFP-RpRalF_189–359 or YFP alone. 24 hours after transfection, cells were lysed and centrifuged at 100,000 g for 1 h. The pellet (M) fraction was resuspended in a volume identical to the supernatant (C) fraction. Samples were blotted with GFP, calnexin (membrane marker) or α-tubulin (cytosol marker) antibodies. <b>B</b>) Protein-lipid overlay assay. The binding of MBP-LpRalF and RpRalF capping domains to indicated lipids immobilized on nitrocellulose membranes was analyzed using an anti-MBP antibody.