Tbk1 nak d1b4

For tissue microarray, we reviewed all H&E-stained slides and representative histological areas were carefully selected and marked on paraffin blocks. From each paraffin block, four primary gastric cancer tissue cores (diameter = 0.6 mm) were taken from the invasive front, both lateral sides, and the luminal surface area of the tumor using AccuMax (IsuAbxis, Seoul, Korea) as previously described [22 (link)]. Immunohistochemistry was performed on formalin-fixed, paraffin-embedded, 4-μm-thick tissue sections using rabbit monoclonal antibody IKKε (D20G4, Cell Signaling Technology, Danvers, MA, USA, 1:50 dilution) and TBK1/NAK (D1B4, Cell Signaling Technology, Danvers, MA, USA, 1:200 dilution). For IKKε, we incubated primary antibody overnight at 4°C and used a DAKO Envision™ Detection Kit (DAKO, Glostrup, Denmark) for 30 minutes. For TBK1, we incubated primary antibody for 15 minutes with Bond-max autoimmunostainer (Leica Biosystem, Melbourne, Australia) using Bond™ Polymer refine detection (DS9800, Vision Biosystems, Melbourne, Australia) according to the manufacturer’s protocol. For the interpretation of IKKε and TBK1 Immunohistochemistry, strong, distinct cytoplasmic staining with membranous accentuation was considered positive.

The proteins derived from 4T1 tumor tissues were extracted and further quantified by the BCA kit. Subsequently, the samples with equal amounts of proteins (20 μg) were separated through SDS-PAGE gel. Then the PVDF membranes with transferred proteins were blocked by 5% skim milk. After incubation with primary antibodies, including phospho-IRF-3 (Ser396) (4D4G) (4947 S, Cell Signaling Technology, 1:1000 dilution), IRF-3 (D83B9) (4302 S, Cell Signaling Technology, 1:1000 dilution), phospho-TBK1/NAK (Ser172) (D52C2) (5483 S, Cell Signaling Technology, 1:1000 dilution), TBK1/NAK (D1B4) (3504 T, Cell Signaling Technology, 1:1000 dilution), phospho-STING (Ser365) (D8F4W) (72971 S, Cell Signaling Technology, 1:1000 dilution), STING (A21051, ABclonal, 1:10000 dilution), and Tubulin β (bs-20694R, Bioss, 1:1000 dilution) overnight at 4 °C, the PVDF films were incubated with anti-rabbit IgG, HRP-linked antibody (7074 S, Cell Signaling Technology, 1:2000 dilution) for another 1 h. Chemiluminescence detection was carried out for protein band visualization with ECL Substrate. Uncropped and unprocessed full scan images of all western blots can be found in the Source Data file.