L α phosphatidylinositol 4 5 bisphosphate pip2

Rapamycin ready-made solution (2.74 mM in DMSO) and Acetylcholine (ACh) chloride were purchased from Sigma-Aldrich (St. Louis, MO, USA). ACh was dissolved in water to make a stock solution of 25 mM. The stock solutions were diluted in bath solution to the final concentrations required for patch-clamp recordings. L-α-phosphatidylinositol-4,5-bisphosphate (PIP₂, natural from porcine brain) was purchased from Avanti Polar Lipids, (Alabaster, AL, USA) and prepared as previously described^{34 (link)}.

1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), l-α-phosphatidylinositol 4,5-bisphosphate (PIP2) (brain, ammonium salt), 1,2-dioleoylphosphatidylinositol 3,4,5-trisphosphate (PIP3) (ammonium salt), 1,2-dioleoyl-sn-glycero-3-phospho-l-serine (DOPS) and extruder accessories were purchased from Avanti Polar Lipids (Alabaster, AL). Texas Red® 1,2-Dihexadecanoyl-sn-Glycero-3-Phosphoethanolamine, Triethylammonium Salt (TR-DHPE) was purchased from Invitrogen (Carlsbad, CA). Calcium- and magnesium-free 150 mM NaCl and 20 mM HEPES buffer (pH = 7.4) were used in the preparation of the vesicle dispersion. Lipid stock solutions of desired compositions were prepared in chloroform and methanol (3:1, v/v) and stored in amber glass vials to protect them from UV light. For each experiment, phospholipids were spread on the walls of a round-bottomed flask, and evacuated in a desiccator for at least two hours to produce an even lipid film. The lipid film was rehydrated, sonicated for 40 min, freeze-thawed 4 times and then extruded at room temperature 17 times through a polycarbonate filter with 50 nm pores. The resulting small unilamellar vesicles (SUVs) were stored at 4°C.

Large unilamellar vesicles were prepared as described before [11 (link), 12 (link)]. Briefly, L-α-phosphatidylcholin (PC), L-α-phosphatidylethanolamine (PE), L-α-phosphatidylserine (PS) and L-α-Phosphatidylinositol-4,5-bisphosphate (PIP₂) were obtained from Avanti Polar Lipids (Alabaster, AL).
To observe the protein-induced deformation of LUVs (as described before [13 (link), 14 (link)]), liposomes (0.2 mg/ml) were incubated with ENTH domain or it’s mutants (15 µM) at 30 °C for 3 h. The samples were subsequently diluted to 0.2 mg/ml of liposomes with HEPES buffer (200 mM NaCl, 10 mM HEPES/NaOH, pH 7.4) and 5 µl of the suspension was then transferred onto a formvar carbon coated copper grid (Agar Scientific Ltd., Essex, UK) and incubated for 1 min at room temperature. The suspension was removed and the grid was set onto a droplet (50 µl) of 3% uranyl acetate for negative staining.
Electron microscopic visualization was performed with a JEOL JEM 1011 transmission electron microscope (JEOL Ltd., Akishima, Japan) and a Gatan Orius 1000 CCS detector (Gatan Inc., Pleasanton, USA).