Solid phase lipid-binding assay, PA bead pull-down assay, and liposome assay

His-PtcC fusion proteins expressed in bacteria were purified by His GraviTrap (GE Healthcare), then eluted with 5 volumes of elution buffer (50 mM Tris HCl, 300 mM NaCl, 500 mM Imidazole, pH 6.8) at 4°C. HA-Ptc and mutants were expressed in S2 cells and then immunoprecipitated with anti-HA antibody and protein A ultralink resin, elution was performed twice in 5 volumes of HA peptide elution buffer (50mM HEPES pH7.9, 100mM NaCl, 1.5 mM MgCl₂, 0.05%Triton X-100, 300 μg/ml HA peptide). Myc-tagged Smo proteins expressed in S2 cells were immunoprecipitated with anti-Myc antibody combined with beads of protein A ultralink resin, followed by two sequential elution with Myc peptide (Abmart, 100 μg/ml Myc peptide, 50 mM Tris HCl,150 mM NaCl, pH 7.4). The eluted purified proteins were concentrated by the Centrifugal filter units (Millipore), followed by dialyzed overnight at 4°C in 25 mM Tris HCl pH7.4, 125 mM KCl, 10% glycerol, 1mM DTT.

Solid phase lipid-binding assay was carried out according to the method Adachi et al. (83). Briefly, all lipids were first solved in chloroform: methanol: H2O (1:1:0.1) at 50 μM, 2 μl of each lipid was spotted onto PVDF membrane, membranes were dried at room temperature and then blocked in PBST (PBS + 1% Tween 20) with 3% fatty acid free BSA for 1 h. 1 μg/ml His-PtcC was then incubated with membrane in PBS overnight at 4°C. Membrane was then washed with PBST (PBS + 0.05% Tween 20) 3 times, followed by Western blotting with the anti-His (ThermoFisher), anti-HA (Santa Cruz) or anti-Myc (Santa Cruz) antibodies. Lipids used in this assay were DOPA ( Avanti Polar Lipids,1,2-dioleoyl-sn-glycero-3-phosphate (sodium salt)), DSPA (Cayman Chemical,1,2-Distearoyl-sn-glycero-3-phosphate (sodium salt)), DPPA (Avanti Polar Lipids,1,2-dipalmitoyl-sn-glycero-3-phosphate (sodium salt)), DMPA (Echelon Biosciences, 1,2-Dimyristoyl-sn-glycero-3-phosphate, sodium salt), DOPC (Cayman Chemical,1,2-Dioleoyl-sn-glycero-3-Phosphocholine), POPE (Cayman Chemical,1-Palmitoyl-3-oleoyl-sn-glycero-2-Phosphoethanolamine), DOPE (Cayman Chemical,1,2-Dioleoyl-sn-glycero-3-Phosphoethanolamine), DPPC (Cayman Chemical,1,2-Dipalmitoyl-sn-glycero-3-Phosphatidylcholine), DOPS (Cayman Chemical,1,2-Dioctadecenoyl-sn-glycero-3-Phosphoserine), DAG (Echelon Biosciences, Dipalmitoyl-sn-glycerol), DPPS (Cayman Chemical,1,2-Dipalmitoyl-sn-glycero-3-phospho-L-serine), PI (Cayman Chemical, PtdIns-(1,2-dioctanoyl) (sodium salt)), PI4P (Cayman Chemical, PtdIns-(4)-P1 (1,2-dioctanoyl) (ammonium salt)).

PA bead pull-down experiments were performed according to Echelon Biosciences Lipid Bead-Protein Pull-down Protocol. Briefly, 20 μg protein was incubated with 50 μl PA beads (Echelon cat #P-B0PA) for 4 h at 4°C, then gently washed twice with 10× wash/binding buffer (10mM HEPES, pH 7.4, 150mM NaCl, 0.25% Igepal). Laemmli sample buffer (50 μl 2× stock solution) was added to the PA beads and the samples were heated to 95°C for 5 mins. Samples were then loaded into SDS-PAGE for Western blot analysis.

Liposomes were prepared according to the methods described by Putta et al. (84). Each liposome contains 400 nmol lipids, control liposomes contain 55 mol% DOPC and 45 mol% POPE; liposomes with DPPA contain 45 mol% DOPC, 45 mol% POPE and 10mol% DPPA. DOPC and POPE were stocked in chloroform at 10mg/ml, DPPA was stocked in chloroform: methanol 1:1 at 1 mg/ml. Lipids were mixed in a glass tube then dried under nitrogen. After that, lipids were suspended in 200 μl freshly prepared extrusion buffer (250 mM Raffinose pentahydrate, 25 mM Tris–HCl pH 7.5, and 1 mM DTT) with occasional vertexing and brief sonication, and then left at room temperature for 40 min to hydrate. Lipids were then diluted with 3 volumes of freshly prepared 1× binding buffer (150 mM KCl, 25 mM Tris–HCl pH7.5, 1 mM DTT, and 0.5 mM EDTA), then centrifuged at 21000g for 45 min to collect liposomes. His-PtcC (0.5 μM) or Myc-Smo (1 μM) was incubated with liposomes at room temperature for 40 min in 100 μl binding buffer. Liposomes were collected by centrifuged at 16000g for 30 min, then washed once with 300 μl binding buffer and transferred into a new tube, centrifuged at 16000g for 30 min to pellet the liposomes. Liposomes were resuspended in 30 μl of 1 × Laemmli sample buffer, heated to 95 °C for 5 min before running on SDS-PAGE.