Beyoecl plus a and b

Three independent mice from each of the infection groups were perfused intracardially with PBS after anesthesia at the indicated time point. Brain tissues were harvested and homogenized on ice using RIPA buffer (Beyotime, Shanghai, China). The lysates were centrifuged at 12,000 rpm for 30 min, and the supernatants were harvested or stored at −80°C. Proteins were separated by 12–15% SDS-PAGE, and target proteins were electroblotted onto PVDF membranes and incubated with antibody against Occludin, Claudin-5, or β-actin. After a second incubation with goat anti-mouse or goat anti-rabbit antibody conjugated with HRP, the membranes were stained with BeyoECL Plus A and B (Beyotime, Shanghai, China) according to the manufacturer’s instructions. Protein fingerprints were shown using Fine-do x6 (Tanon, Shanghai, China). The expression levels of TJ proteins were analyzed by quantifying integrated optical densities (IODs) using Image J v1.51 and normalized by house-keeping gene β-actin.

NA cells cultured in 6-well plates were infected with different viruses at an MOI of 0.1. After incubation at 37 °C for 24 and 72 h, protein samples were harvested to investigate the expression of proteins RAV N, RABV M, RABV G, and GFP in vitro. Briefly, NA cells were washed three times with PBS after the discarding of culture medium and then lysed on ice using RIPA buffer (Beyotian, China). The lysates were centrifuged at 13,000 rpm for 20 min and all supernatants were collected. Proteins were separated by SDS-PAGE, and subsequently transferred on to PVDF membranes and incubated with antibodies against RABV N, RABV M, RABV G, GFP, or β-actin. After a second incubation with goat anti-mouse antibody labeled with horseradish peroxidase (HRP), the membranes were stained with BeyoECL Plus A and B (Beyotime, China) according to the manufacturer’s instructions. Protein fingerprints were shown using Fine-do ×6 (Tanon, China).