Investigating miR-3470b Regulation of BEFV

BHK-21 cells were inoculated with 0.01MOI BEFV after transfection with 50 nM of miR-3470b mimic, NC mimic, miR-3470b inhibitor and NC inhibitor respectively at the indicated time points. After three times freeze-thaw, cells and culture medium were harvested for virus titration expressed as the lgTCID₅₀/ml by the Reed-Muench endpoint method. Meanwhile, BEFV N gene mRNA levels were determined by real-time RT-PCR assay [39 (link)]. All samples were run in triplicate and fold changes were calculated using the 2^-ΔΔCt method. What’s more, BEFV G protein levels were analyzed by western blot as described above using the prepared rabbit anti-G1 polyclonal antibodies with 1/128 virus neutralization titer. Briefly, BEFV infected cells were lysed, and total proteins were subjected to western blot probed with rabbit anti-G1 polyclonal antibodies at a 1: 500 dilution or an anti-β-actin antibody at a 1: 5000 dilution (Abcam, USA). Then, HRP-conjugated anti-rabbit IgG (Jackson, USA) at a 1:3000 dilution was used to label primary antibody. Immunolabelled proteins were visualized using ECL reagent (Pierce).