Rabbit anti ha pab

The protein samples were subjected to 12% SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane. The membranes were blocked with 5% (w/v) skim milk in Tris-buffered saline (TBS) for 2 h at room temperature and then incubated at 4 °C overnight with the following primary antibodies: mouse anti-Flag mAb, mouse anti-actin mAb, rabbit anti-Flag pAb (Proteintech, Wuhan, China), rabbit anti-HA pAb, mouse anti-HN mAb or rabbit anti-NP pAb. After washing three times with TBST, the membrane was incubated for 1 h at room temperature with HRP-conjugated affinipure goat anti-mouse IgG (H + L) or goat anti-rabbit IgG (H + L) (Proteintech, Wuhan, China) diluted 1:3000. ECL Western blot detection reagents were used to visualize the target signals.

Polyinosinic-polycytidylic acid [poly(I:C)] used as the positive control was purchased from InvivoGen (San Diego, CA). The Dual-Luciferase® Reporter Assay System was purchased from Promega (Madison, WI). Mouse anti-SADS-CoV N polyclonal antibody (pAb) [1:1000 for indirect-immunofluorescence assay (IFA), 1:1000 for Western blot (WB), and 1:200 for Confocal] was prepared by our laboratory. Mouse anti-GFP monoclonal antibody (mAb) (1:1000 for WB, and 1:200 for confocal), and rabbit anti-IRF1 pAb (1:1000 for WB, and 1:500 for confocal) were purchased from Santa Cruz Biotechnology (Dallas, TX). Mouse anti-HA mAb (1:3000 for WB, and 1:50 for Co-IP) was purchased from Abmart (Berkeley Heights, NJ). Mouse anti-GAPDH mAb (1:1000 for WB), rabbit anti-HA pAb (4 μg for IP), fluorescein (FITC)–conjugated goat anti-mouse IgG, and horseradish peroxidase (HRP)-conjugated goat anti-mouse/rabbit IgG were purchased from Proteintech (Rosemont, IL, USA). Mouse anti-FLAG mAb, Alexa Fluor 594-conjugated (goat anti-rabbit), Alexa Fluor 488/647-conjugated (goat anti-mouse) secondary antibodies, and MG132 were purchased from Sigma (St. Louis, MO, USA). Chloroquine (CQ) was purchased from MedChemExpress (MCE, Monmouth Junction, NJ, USA). Cycloheximide (CHX) was purchased from Cayman Chemical (Ann Arbor, MI, USA).