Anti strep tag

Anti-SARS-CoV-2 Spike antibody, anti-nucleocapsid antibody, and anti-nucleoprotein antibody were from ProSci, Inc. (Poway, CA, USA). Anti-SARS-CoV-2 anti-Membrane (M) glycoprotein antibody was a custom synthesized rabbit polyclonal antibody to the peptide: KLNDTHSSSSDNIALLVQ (Thermo Fisher Scientific/Invitrogen, Waltham, MA, USA). Anti-TGN46, giantin, GM130, and calnexin were from Abcam (Cambridge, UK). Anti-TGN46 from Thermo Fisher Scientific/Invitrogen, Waltham, MA, USA was used in some experiments. Anti-Strep tag was from Sigma. Monoclonal antibody SCICONS J2 to dsRNA was from English and Scientific Consulting, Kft, Hungary. Secondary antibodies, anti-mouse Ig or anti-rabbit Ig, were Fab’-fragments conjugated to DyLight₄₈₈ or DyLight₅₉₄ or horseradish peroxidase (Jackson ImmunoResearch, West Grove, PA, USA). All primary antibodies and fluorescent secondary antibodies were used at a dilution of 1:200. Anti-horseradish peroxidase secondary antibody was used at 1:100.
Helix pomatia agglutinin (HPA) lectin conjugated to AlexaFluor 488 was from Invitrogen and used at a final concentration of 5 μg/mL. Vital staining with C6-NBD-ceramide (Thermo Fisher Scientific/ Invitrogen, Waltham, MA, USA) was performed as previously described [15 (link)].

The following antibodies were used in the study: anti-Strep Tag (Sigma, SAB2702216), anti-FLAG (Sigma, F1804), anti-V5 (Invitrogen, MA5-15253), anti-IRF3 (Cell Signaling, 4302S), anti-HDAC2 (Abcam, ab124974), anti-Tubulin (Sigma, T5326), anti-STAT1 (Cell Signaling, 14994), anti-STAT1-P (Cell Signaling, 9167), anti-IRF3-S386-P (Abcam, ab76493), and anti-IRF3-S396-P (Cell Signaling, 4947S). Human IFNβ was from Peprotech (300-02BC). The HDAC2 inhibitor Santacruzamat A was from Selleckchem.com (S7595). The HDAC2 siRNA was purchased from Santa Cruz Biotechnology (sc-29345). Lipofectamine RNAiMAX (Invitrogen) was used for the siRNA transfections, which were performed according to the manufacturer’s instructions. 3×FLAG-NSP5 was expressed from the pCDH-CMV-MCS-EF1-Puro vector. The expression plasmids for 2×Strep tagged NSP5, NSP9, NSP10, and EGFP were purchased from Addgene. FLAG-IRF3 and IFNβ-Luc plasmids were gifts from Katherine A. Fitzgerald (UMass Medical School) and Zhijian Chen (UT Southwestern), respectively. The ISRE-Luc reporter vector and the HA-MeV-V expression plasmid were provided by Takeshi Saito (University of Southern California) and Michaela Gack (Cleveland Clinic Florida Research & Innovation Center), respectively.

The following antibodies were used in the study: anti-Strep Tag (Sigma, SAB2702216), anti-FLAG (Sigma, F1804), anti-V5 (Invitrogen, MA5-15253), anti-IRF3 (Cell Signaling, 4302S), anti-HDAC2 (Abcam, ab124974), anti-Tubulin (Sigma, T5326), anti-STAT1 (Cell Signaling, 14994), anti-STAT1-P (Cell Signaling, 9167), anti-IRF3-S386-P (Abcam, ab76493), and anti-IRF3-S396-P (Cell Signaling, 4947S). Human IFNβ was from Peprotech (300-02BC). The HDAC2 inhibitor Santacruzamat A was from Selleckchem.com (S7595). The HDAC2 siRNA was purchased from Santa Cruz Biotechnology (sc-29345). Lipofectamine RNAiMAX (Invitrogen) was used for the siRNA transfections, which were performed according to the manufacturer’s instructions. 3×FLAG-NSP5 was expressed from the pCDH-CMV-MCS-EF1-Puro vector. The expression plasmids for 2×Strep tagged NSP5, NSP9, NSP10, and EGFP were purchased from Addgene. FLAG-IRF3 and IFNβ-Luc plasmids were gifts from Katherine A. Fitzgerald (UMass Medical School) and Zhijian Chen (UT Southwestern), respectively. The ISRE-Luc reporter vector and the HA-MeV-V expression plasmid were provided by Takeshi Saito (University of Southern California) and Michaela Gack (Cleveland Clinic Florida Research & Innovation Center), respectively.