Isolation and Characterization of ER-Derived GUVs

Isolation of the ER-containing membrane fractions from Bcl-2-overexpressing WEHI7.2 cells and preparation of the giant unilamellar vesicles (GUVs) were carried out using OMD patch-clamp technique as described previously [30 (link)]. GUVs were prepared from the 1:5 mixtures of the ER-containing fraction with a 10:1 diphytanoylphosphatidylcholine/cholesterol lipid combination (5 mmol/L). The patch-clamp experiments were carried out using Axopatch 200B amplifier and pCLAMP 10.0 software (Molecular Devices, Union City, CA) for data acquisition and analysis. Patch pipettes were fabricated from borosilicate glass capillaries (World Precision Instruments, Inc., Sarasota, FL) on a horizontal puller (Sutter Instrument Company, Novato, CA) and had a resistance in the range of 7–10 mΩ. Prepared vesicles were immersed in a bath solution containing 150 mmol/L cesium chloride (CsCl), 10 mmol/L HEPES, 1 mmol/L MgCl₂, 2 μmol/L free CaCl₂ [0.9 mmol/L CaCl₂ + 1 mmol/L ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTÅ)], pH 7.2. Patch pipettes were filled with the same solution.