PIP5Ks interact with anionic phospholipids. (a) Purified GST or GST fusion proteins of PIP5K-α and -γ87 were incubated with immobilized phospholipids and detected by immunoblotting using anti-GST antibodies. Chol, cholesterol; SM, sphingomyelin; TAG, triacylglycerol; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; CL, cardiolipin. Results are representative of three independent experiments. (b) Binding of PIP5K-α to lipid-coated beads. GFP–PIP5K-α, partially purified from HeLa cell extracts, was added to C18 Nucleosil beads coated with PC alone (i), 20% PS + 80% PC (ii), 20% PS + 2% PA + 2% PI4,5P₂ + 76% PC (iii), 0.5% PI4P + 99.5% PC (iv), 5% PI4P + 95% PC (v), or 0.5% PI4P + 20% PS + 2% PA + 2% PI4,5P₂ + 75.5% PC (vi). Binding was assessed measuring the green fluorescence associated with the beads. Data are expressed relative to PC-only beads and are means ± SEM of three separate experiments.