Whole-cell extract preparation and immunoblot

Cells were either lysed in 3 packed cell volume (PCV) of radioimmunoprecipitation assay buffer [150 mM NaCl, 50 mM tris-HCl (pH 8), 0.5% sodium deoxycholate, 0.1% SDS, and 1% NP-40] supplemented with 0.5 mM PMSF for 15 min at 4°C or 5 PCV of TENTG-150 for 30 min at 4°C. Lysates were centrifuged 30 min at 13,000 rpm, and supernatants were collected for immunoblot. For phosphorylated protein analysis, buffer was supplemented with PhosphoSTOP (Roche) before whole-cell extraction. Protein quantification was performed using Bradford assay. Samples were resolved on SDS-PAGE, and proteins were transferred onto nitrocellulose membranes. Primary antibodies used include anti-phospho IRF3 (1:500; Cell Signaling 4D4G; 1:500; Abcam Ab76493), anti-IRF3 (1:1000; Cell Signaling D6I4C), anti-phospho TBK1 (1:1000; Cell Signaling D52C2), anti-TBK1 (1:1000; Cell Signaling D1B4), anti-STING (1:1000; Cell Signaling D2P2F), anti-HA (1:10000; Roche), anti–glyceraldehyde-phosphate dehydrogenase (GAPDH; 1:5000; Proteintech Europe 800004-1-Ig), anti-AMP1 (1:1000; Bethyl Laboratories A304896A-M), anti-DARS (1:1000; Bethyl Laboratories A304799A-M), anti-human LysRS (1:1000; Bethyl Laboratories A300630A-M), anti-mouse LysRS (1:2000; Proteintech Europe 14951-1AP), anti-GST (1:10000; Bethyl Laboratories A190122A), anti PARP-1 (1:500; Santa Cruz Biotechnology F-2 sc8007), anti-tubulin (1:2000; Proteintech Europe 66031-1-Ig), anti–TREX-1 (1:250; Santa Cruz Biotechnology C-11 sc133112), and anti-cGAS mouse specific (1:1000; Cell Signaling D3080). The S9.6 antibody was a gift from S. Leppla. All secondary antibodies (Cell Signaling) were used at 1:2000 dilution. Signal was visualized with SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific), and images were acquired on a ChemiDoc (Bio-Rad).