P irf3

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.