P tbk1

For the generation of bone marrow-derived dendritic cell (BMDCs), the bone marrow cells (5 million cells in each 15 cm cell culture dish) were isolated from WT or STING^Gt/Gt mice and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), in the presence of 30 ng/ml GM-CSF (BioLegend), for 10-12 days.
For cytokine assays, BMDCs or B16-F10 melanoma cells were infected with various viruses at an MOI of 10 for 1 hour, or mock-infected. The inoculum was removed, and the cells were washed with PBS two times and incubated with a fresh medium. Supernatants were collected at various times postinfection. Cytokine levels were measured by using ELISA kits for murine IFN-α/β (PBL Biomedical Laboratories), IL-6, CCL5, CXCL10, or GM-CSF (R & D Systems).
For western blot analysis, BMDCs or B16-F10 cells were infected with the indicated viruses at an MOI of 10, and cell lysates were collected at different time points after virus infection. Polypeptides were separated by 15% SDS-PAGE, and western blot analysis was performed to determine the expression of mGM-CSF, using an anti-mGM-CSF antibody (Thermo Fisher), or to investigate the activation status of different components of the cGAS/STING pathway using antibodies against TBK-1, p-TBK-1, IRF-3, p-IRF-3, STING, p-STING, and cGAS (Cell Signaling Technology). GAPDH (Cell Signaling Technology) was used as a loading control.

Protein lysates were prepared as previously described and resolved on 10% SDS–polyacrylamide gels (3 (link), 4 (link)). Following dry transfer, polyvinylidene difluoride membranes were blocked for 1 hour in intercept blocking buffer (LI-COR Biosciences, #927-70001) and incubated overnight in primary antibodies at 4°C. The next day, blots were incubated with fluorescent-conjugated secondary antibodies (LI-COR Biosciences, #926-32221 and #926-32210) for 1 hour. Signals were detected using the Odyssey Infrared Imaging System (LI-COR Biosciences). Actin was used as a loading control.
Primary antibodies for Western blot include pHCK (Abcam, #61055), HCK (Santa Cruz Biotechnology, # N-30), pLYN (Cell Signaling Technology, #2731), LYN (Cell Signaling Technology, #2732), pSRC (Cell Signaling Technology, #2101), SRC (Cell Signaling Technology, #2109), IκBα (Cell Signaling Technology, #9242), pTBK1 (Cell Signaling Technology, #5483), TBK1 (Cell Signaling Technology, #3504), pp65 (Cell Signaling Technology, #3033), p65 (Cell Signaling Technology, #8242), and Actin (Sigma-Aldrich, #A228 for mouse and Cell Signaling Technology, #3700 for human samples).