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

Wang, Yuan; Li, Yan; Ding, Tianbing

We thoroughly mixed 160 µg membrane protein extracts with purified JEV (5x10 6 PFU/ml) in 1 ml microcentrifuge tubes on ice and added an equal volume of 2X IP buffer (150 mM NaCl, 50 mM Tris-HCl, 5 mM EDTA, pH 8.0) containing 1% NP-40 overnight at 4˚C. The complex was incubated with mouse anti-JEV monoclonal antibody (mAb) 2H4 (created and produced in our laboratory) (27) overnight at 4˚C and separated through reacting with protein A/G agarose beads (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) for 2 h at 4˚C in a rocker on the lowest setting. The beads were washed 3 times with 1X IP buffer and centrifuged at 3,000 x g for 5 min at 4˚C, and the pellets with bound proteins were analysed through 10% SDS-PAGE after boiling for 5 min. The protein bands in the gels were stained by Coomassie brilliant blue (Amresco, Solon, OH, USA).