*2.6. Immunofluorescence Staining*

Marc-145 cells were grown on coverslips and then infected with PRRSV at 37 ◦C for 1 h. Cells were washed with PBS after infection and cultured with or without Rg1 (200 μM) for 24 h. Following Rg1 treatment, the cells were fixed with paraformaldehyde for 30 min at 4 ◦C. The fixed cells were permeabilized with 0.1% Triton X-100 in PBS for 5 min, blocked with 3% bovine serum albumin in PBS for 2 h, and endogenous proteins were directly stained with the respective antibodies. For IFA, mouse anti-N protein antibody and goat anti-mouse IgG Alexa Fluor 488 were used as primary and secondary antibodies respectively. Mouse monoclonal antibodies directed against P65 and goat anti-mouse IgG Alexa Fluor 594 were used in confocal assay. The nuclei were stained with 4- ,6-diamidino-2-phenylindole (DAPI). Immunofluorescence was captured by Leica DMI 4000B fluorescence microscope (Leica, Wetzlar, Germany). The cover slips were mounted onto glass slides

using PBS containing 50% glycerol. Confocal images were obtained using a laser scanning confocal microscope (Olympus, Japan).

#### *2.7. Western Blotting*

The total cell samples were washed twice with ice-cold PBS and then lysed in RIPA lysis buffer (Beyotime) with 1% phosphatase inhibitor cocktail (APExBIO, Houston, USA). The protein samples were resolved with sodium dodecyl sulfate–10% polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The PVDF membranes were blocked with 5% BSA in Tris-buffered saline containing Tween 20 and then incubated with the primary antibodies. Goat anti-mouse or anti-rabbit IgG (LI-COR Biosciences) were used as the secondary antibodies. An Odyssey Infrared Imaging System (LICOR, CT, USA) was used to analyze the PVDF membranes.
