BV-2 Cell Infection and Protein Analysis

Unless otherwise stated, BV-2 cells were infected with MNV at an m.o.i. of 5 TCID₅₀ per cell, and 12 h post-infection the cells were harvested and lysed in RIPA buffer [50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1 % Triton X-100, 0.1 % SDS]. Cell lysates were resolved by SDS-PAGE and then probed using anti-GAPDH (AM4300; Ambion), anti-NS7, anti-ATP5α (ab14748; Abcam), or a mouse monoclonal anti-VF1 (4K5; Abmart) antibodies. The anti-NS7 antibody was described previously [24 (link)]. Goat anti-rabbit (sc-2030; Santa Cruz) and goat anti-mouse (sc-2031; Santa Cruz) secondary antibodies conjugated with HRP were used for detection by chemiluminescence using the Amersham ECL Prime Kit (GE Healthcare). Biochemical fractionation of infected cells was performed as previously described [19 (link)].