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

Wang, Yuan; Li, Yan; Ding, Tianbing

Western blot analysis for HSP90β-JEV binding. We routinely used western blot analysis to detect JEV binding with HSP90β from the Vero membrane protein extracts under non-reducing and non-boiling conditions. Due to the unavailability of anti-monkey HSP90β antibodies and the high homogeneity of HSP90β between humans and monkeys, a rabbit anti-human HSP90β mAb (Product no. ab32568; Abcam, Cambridge, UK) was used for blotting (1:500 in TBS containing 5% powdered milk). We then used Odyssey™ IRDye680-labelled goat anti-rabbit IgG (1:5,000; Cat. no. 926-68071; LI-COR Biosciences, Lincoln, NE, USA). In a parallel blotting test, rabbit anti-human HSP90β mAb was replaced by purified JEV (1x10 9 PFU/ml) followed by the addition of anti-JEV mAb 2H4 (22 µg/ml) and Odyssey™ IRDye680-labelled goat anti-mouse IgG (1:5,000; Cat. no. 926-68070; LI-COR Biosciences). The results were scanned and analysed by GelDoc-It™ (Ultra-Violet Products Ltd., Cambridge, UK).