Protein-lipid overlay assay

A lipid strip was prepared by spotting 100 pM of each lipid on a nitrocellulose membrane (0.2 μm; Bio-Rad, Hercules, CA, USA). 1,2-didecanoyl-sn-glycero-3-phosphorylated-l-serine (PS), PC (1,2-dioleoyl-sn-glycero-3-phosphocholine), PA (1,2-dioleoyl-sn-glycero-3-phosphate), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (PE), and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] were obtained from Avanti Polar Lipids. Phosphatidylinositol 3, 4, 5-trisphosphate was obtained from Matreya (State College, PA, USA). D-(+)-sn-1-O-oleoyl-glyceryl-3-phosphate, sphingosine 1-phosphate, phosphatidylinositol diC16, phosphatidylinositol 3-phosphate diC16, phosphatidylinositol 3,4-bisphosphate diC16, phosphatidylinositol 3,5-bisphosphate diC16, phosphatidylinositol 4-phosphate diC16, phosphatidylinositol 5-phosphate diC16, and 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine were obtained from Echelon. The strips were blocked with 5% fatty acid–free bovine serum albumin in Tris-buffered saline with Tween for 2 h, followed by incubation with 1 μg/ml GST-IQGAP1 proteins in blocking buffer overnight. The lipid strips were then incubated with a rabbit anti-GST antibody for 1 h and a horseradish peroxidase–conjugated goat anti-rabbit IgG for 1 h. The strips were washed with Tris-buffered saline with Tween for 5 min, 3 times each. The lipid-bound protein was visualized with exposure to X-ray film using an ECL Kit from Thermo Fisher Scientific.