Characterization of in vitro FGF2 membrane recruitment assays using a FGF2-GFP variant form impaired in binding to PI(4,5)P2. Quantitative comparison of PI(4,5)P₂-dependent binding efficiencies of FGF2-WT-GFP and a variant form known to be impaired in membrane recruitment lacking two essential amino acids of the PI(4,5)P₂ binding pocket (FGF2-K127Q/R128Q-GFP [FGF2-K127Q/R128Q-GFP]; Temmerman et al., 2008, Traffic). (A) Liposomes made from a PC/Cholesterol/PI(4,5)P₂ mixture. (B) Liposomes made from a complex or plasma-membrane-like lipid composition. Details on lipid compositions are given in Table S1. Data were acquired after 1 h incubation and corrected for background defined by the binding of GFP to the liposomal systems indicated. (C) Protein analysis by SDS-PAGE and Coomassie staining for all recombinant proteins used in in vitro biochemistry assays. From left to right: His-HaloTag, His-FGF2-WT-HaloTag, His- PH-PLC-δ1-HaloTag, His-GFP, His-FGF2-Y81pCMF-WT-GFP and His-FGF2-Y81pCMF-K127Q/R128Q-GFP. Data are shown as mean ± SEM (n = 3).