(a) TBK1 K38A mutant reconstituted in BJ TBK1/IKKε double knockout (DKO) cells cannot be phosphorylated in response to cGAMP or HT-DNA treatment. BJ wild type and TBK1/IKKε DKO cells reconstituted or not with wild type or K38A TBK1 were stimulated with 100 nM cGAMP for 1 h or transfected with 2 μg/ml HT-DNA or lipofection alone for 3 h. Whole cell lysates were harvested for immunoblotting analysis. Stable re-expression of wild-type (WT) or K38A TBK1-Flag in TBK1/IKKε DKO cells was achieved by lentiviral infection. (b) YM201636 treatment inhibits STING/TBK1 interaction in co-IP. BJ cells stably expressing Flag-STING were pretreated with DMSO or 5 μM YM201636 for 18 h and stimulated with cGAMP for 1 hour, followed by IP of whole cell lysates with anti-Flag M2 affinity gel. The cell lysate and IP samples were analyzed by immunoblotting. (c) After a liposome-based reaction as depicted in Fig. 3a, most TBK1, p-TBK1 and STING were associated with the membrane fraction. After 10 min of reaction at 37 °C, half of sample was saved as total input and the other half was centrifuged at 20,000 xg for 10 min at 4 °C. Pellet and supernatant were diluted with SDS loading buffer to the same volume of the total sample and analyzed by immunoblotting. cGAMP: 1 μM; diC8 PtdIns(3,5)P 2 : 25 μM in solution; ATP: 2 mM. (d) Schematic diagram of TBK1 membrane recruitment assay. (e) PtdIns(3,5)P 2 does not affect cGAMP-induced TBK1 recruitment to STING-containing membranes. STING-containing liposomes derived from Flag-STING expressing COS7 cells were stimulated with 1 μM cGAMP at room temperature for 10 min in the presence or absence of 25 μM diC8 PtdIns(3,5)P 2 in solution. With or without an additional 10 min incubation with 2 mM ATP, the samples were then centrifuged at 20,000 xg for 10 min; pellets (P) and supernatants (S) were subjected to SDS-PAGE and immunoblotting. (f) Depletion of PIKfyve does not affect cGAMP-stimulated TBK1 recruitment to STING-containing membranes. STING-containing liposomes derived from Flag-STING expressing BJ PIKfyve CRISPR cells pretreated with control or PIKfyve siRNA for three days were stimulated and processed as in (e) without the final ATP step. The same concentrations of cGAMP and diC8 PtdIns(3,5)P 2 as in (e) were used. The schematic in d was created in BioRender. Tan, X. (2025) https://biorender.com/w5x5p41.