Cholesterol enhances PI(4,5)P ₂ -dependent membrane recruitment of FGF2 and PH-PLC-δ1 to lipid bilayers. FGF2-Halo-AF488 and PH-PLC-δ1-Halo-AF488 binding to PI(4,5)P₂-containing liposomes were quantified using an analytical flow cytometry assay described previously (Temmerman et al., 2008; Temmerman and Nickel, 2009). (A) Kinetic analysis using liposomes containing 5 mol% PI(4,5)P₂, 30 mol% cholesterol and 65 mol% PC (PC5-CHOL30). Measurements were taken after 1, 3, 6, 12, 18, and 24 h of incubation. (B) Quantitative analysis of FGF2-Halo-AF488 binding to various kinds of liposomes containing different levels of cholesterol after 1 (subpanel a), 3 (subpanel b), 6 (subpanel c), and 24 h (subpanel d) of incubation. FGF2-Halo-AF488 binding to liposomes containing 10 mol% PI(4,5)P₂ and 90 mol% PC (PC10 system; positive control) and liposomes consisting of 100 mol% PC (PC0 system; negative control) were used to normalize data. (C) FGF2 binding to liposomes with a PM-like lipid composition, in the presence (30 mol%) or absence of cholesterol. The data were acquired after 1 h of incubation time. (D) Quantitative analysis of PH-PLC-δ1-Halo-AF488 membrane binding to liposomes containing either 0 or 30 mol% cholesterol after 1 h incubation. PH-PLC-δ1-Halo-AF488 binding to liposomes containing 10 mol% PI(4,5)P₂ and 90 mol% PC (PC10 system; positive control) was used to normalize data. (E) PH-PLC-δ1-Halo-AF488 membrane recruitment assays using PM-like lipid compositions in the presence (30 mol%) or absence of cholesterol. The data were acquired after 1 h of incubation time. All data were corrected for background defined by binding of Halo-AF488 to the various liposomal systems indicated. Standard errors (n = 3 for A, C, and E; n = 6 for B; n = 12 for D) and P values are shown with *P ≤ 0.05; ***P ≤ 0.001. The statistical analysis was based on a one-way ANOVA test combined with Tukey’s post hoc test. Data distribution was assumed to be normal, but this was not formally tested.