Xt sample buffer and reducing agent

Plasmid empty vector (EV) and FLAG-LidI^{PLE 1} were grown to OD_600-tube=0.2 then induced with 1 mM IPTG and 1.5 mM for 40 minutes before 0.5 mL samples were taken. To observe expression during infection, strains were grown to OD_600-tube=0.3, infected with ICP1 MOI = 2 with 4 mL samples taken at the labeled timepoints. Samples were prepared and visualized as previously described (McKitterick and Seed, 2018 (link)). Briefly, samples were mixed with one volume of cold methanol, pelleted at 15,000 x g at 4°C for 15 minutes, washed with 1 mL cold PBS, and pelleted. Pellets were resuspended in PBS with XT sample buffer and reducing agent (Bio-Rad), vortexed, and boiled for 10 minutes before being run on 4–12% Bis-Tris SDS gels (Bio-Rad Criterion XT). Gels were transferred via the Trans-Blot Turbo (Bio-Rad) and visualized with rabbit-α-FLAG (1:3,000) primary and goat-α-rabbit-HRP conjugated secondary antibodies on the ChemiDoc MP Imaging System (Bio-Rad).

Cellular lysates for detection of STING dimerization by western blotting were prepared using non-reducing RIPA lysis buffer with 0.2% SDS and complete mini inhibitor (Sigma). For detection of pTBK1 NaF (10 mM) was added to the lysis buffer. Separation by SDS-page was perfomed using Criterion precast gels (Biorad) and the protein transfer by Biorad Trans-blot turbo system. XT Sample buffer and reducing agent was also purchased from Biorad. For STING dimerization blotting of STING was performed using a mRabbit antibody (D2P2F, dilution 1:1,000) from Cell Signaling Technology. Antibodies for pTBK1 was purchased also from Cell Signaling Technology (dilution 1:1,000). hemagglutinin (dilution 1:1,000) and Giantin (dilution 1:1,000) antibodies were purchased from Abcam. Secondary antibodies for confocal imaging and western blotting were purchased from Invitrogen (dilution 1:500) and Jackson ImmunoResearch (dilution 1:10,000), respectively. Uncropped immunoblots are shown in Supplementary Fig. 9. For confocal imaging cells were permeabilized with ice-cold methanol and then blocked in 1% bovine serum albumin. Cells were then stained with respective antibodies, with DAPI for nuclear staining, mounted in proLong Gold (Molecular probes) and vizualised using a Zeiss LSM 710 laser-scanning microscope.

The following primary antibodies were used in western blot: γH2AX (1:2000 dilution, Millipore, cat# 05–636), GAPDH (1:1000 dilution, Cell signaling, cat# 3683), gamma-tubulin (1:5000, Thermo Scientific cat#MA1-850), c-MYC (1:1000, Cell signaling, cat# 5605), N-MYC (1:1000, Cell signaling, cat# 9405), L-MYC (1:1000, R&D systems, cat# AF4050) and PRKDC (1:200 dilution, Santa Cruz, cat# sc-9501). For immunoblots, cells were lyzed with either CelLytic M cell lysis buffer (Sigma) or RIPA buffer (Sigma cat#89900) and equal amounts of protein lysates were mixed with XT sample buffer and reducing agent (Bio-Rad), separated by SDS Criterion precast gels (Bio-Rad), and transferred to a PVDF membrane (Bio-Rad). Proteins were detected with primary antibodies and horseradish peroxidase-conjugated secondary antibodies by using SuperSignal West Dura Extended Duration Substrate (Thermo Scientific).