Avanti mini extruder

Ten microliters of the lipid mixture (PC:PE, 3:1, or PC:PE:PS, 3:1:1, mol/mol) was dried in glass bottles and rehydrated with 50 mM CF, 100 mM sucrose, and 5 mM HEPES/KOH (pH 7.4). Multilamellar liposomal suspensions were extruded with a 0.1 µm polycarbonate membrane using an Avanti Mini Extruder and purified by using a PD-10 column (GE Healthcare, Amersham, UK). The p7 with or without GdCl₃, LaCl₃, or NN-DNJ was added to LUVs, after which the CF leakage was recorded with a Fluoroskan Ascent FL (Thermo Labsystems, Loughborough, UK) in external buffer (100 mM KCl, 100 mM sorbitol, and 5 mM HEPES/Tris, pH 7). CF leakage was calculated using the following formula: $\mathrm{CF}\mathrm{leakage}(\%)=\frac{\mathrm{F}-{\mathrm{F}}_0}{{\mathrm{F}}_{\max }-{\mathrm{F}}_0}\times 100$
where F = measured fluorescence intensity, F₀ = basal LUVs fluorescence intensity, and F_max = LUVs treated with 0.1% Triton X-100.

LUVs were made by extrusion. Lipid solutions containing 12.5 μmole of lipids were made using a 4:1:0.025 molar ratio of POPC:POPG:Ch or a 5:0.025 molar ratio of POPC:Ch, expect where indicated and as previously described13 (link). In brief, the lipid solution was dried under a stream of argon gas, residual chloroform was then removed by storage for 1 hr under vacuum. Lipid films were then dissolved in 500 μl of potassium buffer (10 mM K₂HPO₄, 50 mM K₂SO₄, 5 mM MOPS, pH 7.4) and hydrated for 30 min at 5 °C above the phase transition temperature of the lipid mix with vortex mixing every 5 min. Lipid solutions were then freeze-thawed 15 times transferring between a dry ice ethanol bath and a water bath at hydration temperatures before extrusion using an Avanti Mini-Extruder, 26 times with 100 nm pore-size membranes (Whatman Nuclepore Track-Etched Membranes, GE Healthcare Bio-Sciences, PA, USA) at hydration temperatures. LUVs were stored at 4 °C and used within one week.

Portions (10 μl) of the lipid mixture of E. coli total lipid extract were dried and rehydrated with 50 mM CF, 100 mM sucrose, and 5 mM HEPES/KOH (pH 7.4). Multilamellar liposomal suspensions were extruded with a 0.1-μm polycarbonate membrane using an Avanti Mini Extruder and purified by a PD-10 column (GE Healthcare, Buckinghamshire, United Kingdom) as previously described. The peptides were added to LUVs, and CF leakage was measured using Fluroskan Ascent FL (Thermo Labsystems, UK) in external buffer (150 mM KCl and 10 mM HEPES/Tris; pH 7). CF leakage was calculated using the following formula: CF leakage (%) = 100 × (F – F₀)/(F_max – F₀), where F is the measured fluorescence intensity, F₀ is the basal LUV fluorescence intensity, and F_max is the fluorescence intensity of LUVs treated with 0.2% Triton X-100.