Heat shock protein 90ß in the Vero cell membrane binds Japanese encephalitis virus

Wang, Yuan; Li, Yan; Ding, Tianbing

Cell membrane protein extraction. Vero cells (ATCC ® CCL-81™) were obtained from Ms. L. Jia (The National Institute for the Control of Pharmaceutical and Biological Products). Vero cell membrane proteins were prepared as previously described (26) with minor modifications. Briefly, the cells were cultured in RPMI-1640 medium containing 10% FCS in an incubator (Hera Cell; Heraeus Holding GmbH, Hanau, Germany) with 5% CO 2 at 37˚C. Confluent cell monolayers were washed 3 times with PBS, treated with 1 mM EDTA in PBS (pH 7.2) for 3 min at 37˚C, and subsequently resuspended in ice cold Buffer M (100 mM NaCl, 20 mM Tris-HCl pH 8.0, 2 mM MgCl 2 , 1 mM EDTA and 1 mM β-mercaptoethanol) containing 1 mM phenylmethanesulfonyl fluoride (PMSF; Sigma-Aldrich, St. Louis, MO, USA) on ice for 40 min with intermittent shaking, and lysed using 15-20 strokes and a Dounce homogeniser. The nuclei and cell debris were removed by centrifugation at 500 x g and 4˚C for 10 min. The membrane-bound proteins in the supernatant fraction were condensed through further centrifugation at 36,000 x g and 4˚C for 30 min. The pellet was dissolved in Buffer M without β-mercaptoethanol. The concentrations of the membrane proteins were determined using a BCA kit (Pierce; Thermo Fisher Scientific Inc., Rockford, IL, USA).