Anti mavs

Sendai virus was purchased from The American Type Culture Collection (ATCC, Manassas, VA). Reovirus was purchased from Advanced Biotechnologies, Inc. (Columbia, MD). Poly I:C, poly dG:dC and biotin-labeled Poly I:C were purchased from InvivoGen (San Diego, CA). Lipofectamine^™ 2000 was purchased from Invitrogen (Carlsbad, CA). DHX15 antibody was purchased from Abcam (Cambridge, MA) and used for immunoprecipitation and immunoblotting. The following antibodies were used for immunoblotting: anti-IRF3 (Santa Cruz, Dallas, TX); anti-DDX21 (Novus Biologicals, Littleton, CO); anti-MAVS, anti-STING, anti-Erk1/2, anti-p38, anti-p65, anti-phospho-Erk1/2, anti-phospho-p38, anti-phospho-p65 and anti-phospho-IRF3 (Cell Signaling, Danvers, MA); anti-DHX41, anti-GAPDH-HRP, anti-Flag-HRP, Anti-HA-HRP and anti-Myc-HRP (Sigma, St. Louis, MO). Anti-HA and anti-Myc beads were purchased from Sigma. Protein A/G beads and NeutAvidin beads were purchased from Thermo Scientific (Rockford, IL).

Hoechst 33342 (Invitrogen) was used at a concentration of 100 ng/ml. MG-132 (Cayman Chemical) was used at 10 μM and 3 Methyladenine (Sigma-Aldrich) at 5 nM. Primary antibodies used for immunofluorescence experiments were the following: anti-MAVS (Cell Signaling, 1:500), anti-Flag (Sigma, 1:500), anti-Tomm20 (Santa Cruz, 1:1,000), and anti-DRP1 (BD Biosciences, 1:200). The secondary antibodies used for immunofluorescence were the following: polyclonal anti-mouse Ig Alexa488 or Alexa568 conjugated (Invitrogen, 1:1,000), polyclonal anti-rabbit Ig Alexa488, Alexa568, or Alexa647 conjugated (Invitrogen, 1:1,000), polyclonal goat anti-rabbit Ig ATTO647N (Active Motif, 1:1,000), and polyclonal goat anti-mouse IgG MegaRed 520 (Sigma, 1:100). Primary antibodies used for immunoblotting were the following: rabbit anti-MAVS, ATG5 and DRP1, mouse anti-GFP, and IFN-α (Cell Signaling), mouse anti-PCPB2 and anti-AIP4 (Santa Cruz Biotechnology), monoclonal anti-Myc (Clontech Laboratories), mouse anti-Flag and rabbit anti-LC3 (Sigma-Aldrich), rabbit anti-K48-linked polyubiquitin chains (Millipore), mouse monoclonal anti-IFN-β (Biolegend), and mouse anti-actin conjugated to horseradish peroxidase (HRP, Sigma-Aldrich). For immunoprecipitations monoclonal mouse anti-Myc, anti-PCPB2 or anti-GFP and Protein G PLUS–Agarose (Santa Cruz Biotechnology) were used.

IFNβ and IFNλ1 were purchased from Biolegend. PAMPs were purchased from InvivoGen. diABZI, ruxolitinib and H-151 were purchased from Selleck Chemicals. The following antibodies were used for Western blotting or immunofluorescence: anti-RIG-I (Cell Signaling Technology, 3743S), anti-MDA5 (Cell Signaling Technology, 5321S), anti-MAVS (Cell Signaling Technology, 3993S), anti-cGAS (Cell Signaling Technology, 83623S), anti-STING (Cell Signaling Technology, 13647S), anti-STING (Proteintech, 19851-1-AP), anti-Phospho-STING (Ser366) (Cell Signaling Technology, 50907S), anti-TBK1 (Cell Signaling Technology, 3504S), anti-Phospho-TBK1 (Ser172) (Cell Signaling Technology, 5483S), anti-IRF3 (Cell Signaling Technology, 11904S), anti-Phospho-IRF3 (Ser396) (Cell Signaling Technology, 4947S), anti-NF-κB p65 (Cell Signaling Technology, 8242S), anti-Phospho-NF-κB p65 (Ser536) (Cell Signaling Technology, 3033S), anti-TRIM22 (Invitrogen, PA5-51964), anti-SARS-CoV-2 nucleocapsid (GeneTex, GTX135357), and anti-tubulin (Sigma, T6199). Anti-SARS-CoV-2 Spike antibody (CR3022) was from Absolute Antibody. HRP-conjugated secondary antibodies (anti-mouse or anti-rabbit) were purchased from Amersham. Alexa Fluor-conjugated secondary antibodies were purchased from Invitrogen.