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Fig. 3. Analysis of recombinant EE-FENS-1, iFYVE FENS-1 and EE-DFCP1 binding to lipid surfaces using BiaCore. (A) Sensorgrams describing the binding of EE-FENS-1 to PC liposomes containing 1 mole% PtdIns3P or 1 mole% PtdIns5P captured on an L1 sensor chip. Binding of EE-FENS-1 to PC alone was low (approx. 20 response units at equilibrium) and this sensorgram has been subtracted from the two shown to remove bulk refractive-index changes that are due to the ethylene-glycol-containing FENS-1 solution. 100 nM FENS-1 diluted into PBS, 1 mm MgCl2 was injected at t=0 for 360 seconds (at 10 µl min-1) and then the dissociation followed for a further 400 seconds. The inset shows a Coomassie-stained SDS PAGE gel of approx. 2 µg of recombinant EE-FENS. (B) Binding of EE-FENS-1 to PC monolayers alone or containing 6 mole% phosphoinositides, assembled on an HPA sensor chip. The values shown are the means±s.e.m. (n=2-4) for the binding of approx. 100 nM EE-FENS-1 at equilibrium. (C) Sensorgram describing the binding of iFYVE FENS-1 to a PE/PC/PS (1:1:1 mole ratios) monolayer containing 1 mole% PtdIns3P assembled on an HPA sensor chip. Binding of iFYVE FENS-1 to PE/PC/PS surfaces only was low (approx 26 response units at equilibrium) and this sensorgram has been subtracted from the one shown to remove bulk refractive index changes caused by the glycerol-containing iFYVE FENS-1 solution. 100 nM iFYVE FENS-1 diluted into PBS, 1 mM MgCl2 was injected at t=0 for 360 seconds (at 10 µl min-1) and then dissociation followed for a further 400 seconds. The inset shows a Coomasie-stained SDS PAGE gel of approx 2 µg thrombin-cleaved, recombinant iFYVE FENS-1. (D) Sensorgrams describing the binding of EE-DFCP1 to PC liposomes alone or containing 1 mole% PtdIns3P or PtdIns5P, captured on an L1 sensor chip. 50 nM EE-DFCP1 diluted into PBS, 1 mM MgCl2 was injected at t=0 for 360 seconds (at 10 µl min-1) then dissociation followed for a further 400 seconds. The instant refractive index changes at the point of injection and dissociation are caused by the bulk refractive index changes to and from the ethylene glycol containing EE-DFCP1 solution. The inset shows a Coomassie-stained SDS PAGE gel of approx 2 µg EE-DFCP1.