Preparation of liposomes and liposome co-sedimentation assay

Liposomes were prepared from chloroform stocks of 10 mg/ml of POPC, 16:0–18:1 and 1 mg/ml of porcine brain L-α-phosphatidylinositol-4,5-bisphosphate, PI(4,5)P₂ (both Avanti Polar Lipids, USA). Lipid mixtures of 0% to 20% of PI(4,5)P₂ and 80% to 100% of POPC were first dried under a stream of nitrogen, and then vacuum dried for at least 1 h. Dried lipid mixtures were hydrated in 20 mM Tris, 200 mM NaCl, 2% glycerol, 0.5 mM DTT, pH 8.5, incubated for 10 min at room temperature, gently mixed, further incubated for 20 min, and sonicated for 2 min at room temperature in a water bath, followed by 5 freezing-thawing cycles. Using a mini-extruder (Avanti Polar Lipids), vesicles were then extruded through polycarbonate filters with 0.4 μm and 0.1 μm pore sizes, respectively. In order to avoid the presence of potential aggregates, the Ig3 domain of palladin, Ig1Ig2^250–444, and Doc2b, a positive control for PI(4,5)P₂ binding, were first centrifuged in a TLA-55 rotor for 30 min at 100,000 × g, 4°C, using an optima MAX-XP benchtop ultracentrifuge (Beckman Coulter Life Sciences, USA). For the assay, proteins and liposomes were mixed in polycarbonate tubes and incubated for 30 min at room temperature. Final concentrations of liposomes and proteins were 0.5 mg/ml and 10 μM, respectively. Mixtures were ultracentrifuged in a TLA-100 rotor for 40 min at 100,000 × g, 20°C. Pellets and supernatants were separated and equal volumes analyzed by SDS-PAGE. Gels were scanned and quantified by densitometry with the QuantiScan 1.5 software (Biosoft).