Preparation of Plasma Membrane Vesicles from CHO Cells.

Enriched plasma membrane fractions were prepared as described elsewhere (Rossi et al., 2012). CHO cells from 10 confluent 175 cm² flasks (Thermo Scientific, Rochester, NY) were homogenized by 20 strokes of a glass Dounce homogenizer in 250 mM sucrose, 10 mM Tris-HCl, and 1 mM EDTA containing protease inhibitors (Complete Mini; Roche Diagnostics, Mannheim, Germany). The homogenate was then centrifuged at 4000g for 15 minutes at 4°C, and the enriched plasma membrane fraction was obtained by centrifugation at 17,000g for 45 minutes. The resultant pellet was suspended in PBS for stopped-flow measurements.