Irdye 800 labeled igg

Vero cells were infected with VACV recombinants expressing SARS-CoV-2 spike protein or RBD. Cell lysates were harvested 24 h post-infection; samples were boiled for 10 min, separated in a 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel, and transferred onto nitrocellulose filter membranes. After being blocked with 5% milk, membranes were probed with the antibodies anti-SARS-CoV-2 S2 (GTX632604, GeneTex, Irvine, CA, USA), anti-SARS-CoV-2 RBD (Sino Biological Inc., Beijing, China; 40592-T62), and anti-actin (Sigma-Aldrich, St. Louis, MO, USA; A1978) followed by incubation with IRDye 800-labeled IgG and IRDye 680-labeled IgG secondary antibodies (Li-Cor Biosciences, Lincoln, NE, USA). Protein signals were scanned with Odyssey (Li-Cor Biosciences, Lincoln, NE, USA).

The 23 genes of SARS-CoV-2 (IPBCAMS-WH-01/2019 strain, no. EPI_ISL_402123) were optimized by Gene Designer 1.0 and cloned to vector pCMV6-entry expression vector with the FLAG-tag or HA-tag at C-terminus. Plasmids Flag-RIG-I, Flag-RIG-IN, Flag-MDA5, HA-MAVS, pGL3-IFN-β–Luc, IRF3-5D-Flag, and pRL-TK have been described elsewhere⁴² . The mutated variants of the SARS-CoV-2 ORF6-tagged Flag were constructed by using a Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA). All variants were confirmed by subsequent sequencing.
The antibodies used in this research were: Flag antibody from Sigma-Aldrich (1:4000, Cat# F3165); β-actin antibody from Sigma–Aldrich (1:4000, Cat# A5441); HA antibody from Sigma–Aldrich (1:10,000, Cat# H9658); STAT1 antibody from Cell Signaling technology (1:1000, Cat# 9172); P-STAT1 antibody from Thermo Fisher (1:1000, Cat# 700349); Sev antibody from MBL (1:2000, PD029C1). Dual-Luciferase® Reporter Assay System was purchased from Progema (Madison, WI). IRDye 800-labeled IgG and IRDye 680-labeled IgG secondary antibodies were purchased from Li-Cor Biosciences (Lincoln, NE).