Tbk1 antibody

The polyclonal antibody against STING was generated by immunizing rabbit with recombinant human STING (221–379 aa). The goat polyclonal antibody against SCAP was from Santa Cruz Biotechnology. The rabbit polyclonal antibody against SCAP was a gift from Dr. Xiongzhong Ruan (Chongqing medical university). hemagglutinin (HA), Myc, Ub, SREBP1/2 and IRF3 antibody were purchased from Santa Cruz Biotechnology. TBK1 antibody was from abcam. Tom20 antibody was from BD Biosciences. Flag and β-actin antibodies were obtained from Sigma-Aldrich. Phospho-IRF3 and Phospho-TBK1 antibody was from Cell Signaling Technology.
Poly(dA:dT) and lipopolysaccharide (LPS) was obtained from Sigma-Aldrich. Poly(I:C) was purchased from Invitrogen. Wild type HSV-1 and HSV-1-GFP were kindly provided by Dr. Wentao Qiao (Nankai University) and Dr. Chunfu Zheng (Suzhou University), respectively. Listeria monocytogenes (10403 serotype) was a gift from Dr. Youcun Qian (Institute of Health Sciences). TBK1 kinase inhibitor BX795 was purchased from InvivoGen. ISD (Interferon stimulatory DNA) was prepared by annealing equimolar amounts of sense and antisense DNA oligonucleotides at 95°C for 10 min before cooling to room temperature. Oligonucleotides used are as follows:

The polyclonal antibody against SENP7 was generated by immunizing rabbit with recombinant mouse SENP7 (716–1010 aa). The rabbit polyclonal antibody against cGAS was obtained by immunizing rabbit with recombinant mouse cGAS (362–643 aa). The antibodies against hemagglutinin (HA), Myc and IRF3 were purchased from Santa Cruz Biotechnology. Mouse monoclonal Flag antibody and β-actin antibodies were obtained from Sigma-Aldrich. The SUMO antibody was from Abcam. Phospho-IRF3 antibody, Phospho-TBK1 antibody, and rabbit DYKDDDDK tag antibody were from Cell Signaling Technology. TBK1 antibody was from Abcam. Anti-Flag (M2)-agarose and EZview red anti-HA affinity gel were from Sigma. Poly(dA:dT) was obtained from Sigma. Interferon stimulatory DNA (ISD) was prepared by annealing equimolar amounts of sense and antisense DNA oligonucleotides at 95°C for 10 min before cooling to room temperature. Oligonucleotides used are as follows: ISD (sense), 5′-TAC AGA TCT ACT AGT GAT CTA TGA CTG ATC TGT ACA TGA TCT ACA-3′; ISD (antisense), 5′-TGT AGA TCA TGT ACA GAT CAG TCA TAG ATC ACT AGT AGA TCT GTA-3′. cGAMP was from InvivoGen and was delivered into cultured cells by digitonin permeabilization method as previously described (Girardin et al., 2003).

Commercially available antibodies used for this study include mouse monoclonal FLAG M2 antibody (1:5000) (Sigma); rabbit polyclonal V5 antibody (1:5000) (Thermo Fisher); mouse monoclonal V5 (1:5000), β-actin (1:5000), and TBK1 antibody (1:1000) (Abcam); Phospho-TBK1 (Ser172) (1:1000), Phospho-IRF3 (Ser396) (1:1000), Ku80 (1:1000), cGAS (D1D3G) (1:1000), and STING (D2P2F) antibody (1:1000) (Cell Signaling); GAPDH (6C5) (1:1000), VDAC1 (B-6) (1:1000), and IRF3 antibody (FL-425) (1:1000) (Santa Cruz); DNA-PKcs (1:1000) and Ku70 antibody (1:1000) (NeoMarkers); and phospho-Histone H2A.X (Ser139) antibody (1:1000) (Millipore).
Rabbit polyclonal anti-cGAS pT68 and pS213 antibodies were generated by GenScript Company (NJ, USA) and used at 1:500 dilution. Rabbit polyclonal anti-MAVS antibody was generated in the laboratory and used at 1:1000 dilution^{48 (link)}.
Nu7441 (Selleck Chemical), Nu7026 (MedChemExpress), Herring testis (HT)-DNA (Sigma-Aldrich), poly I:C (InvivoGen), 2’,3’-cGAMP (InvivoGen), streptavidin agarose (Thermo Fisher), Ni-NTA His-Bind Resin (Novagen), and Malachite Green Phosphate Detection Kit (Cell Signaling) were used according to the manufacturer’s protocol. Biotin-labeled ISD-45 DNA was ordered from IDT. [α-P32]-ATP was ordered from Perkin Elmer. Lipofectamine 2000 was purchased from Invitrogen.