Figure 4. Activated TBK1 stabilizes SIKE1 and SLMAP proteins.

(A–C) Gels and images showing that DNA damage triggered an enhancement of SLMAP-TBK1 interactions. Samples of 293FT cells were (A) transiently transfected with Flag-SLMAP, (B) cotransfected with Flag-TBK1 and HA-SLMAP, or (C) transfected with Flag-TBK1. Cells were treated as indicated and harvested 2 hours after treatment for either the (A and C) co-IP or (B) immunofluorescence assay. Scale bar: 10 μm. (D) Gels showing that cGAS-STING activation enhanced TBK1-SIKE1-SLMAP ternary interactions. 293FT cells transfected with Flag-TBK1 were treated with cGAMP for 1 hour before being processed for an IP assay conducted 2 hours after the treatment. (E) Hypothetical structural model of the SLMAP-SIKE1-TBK1 ternary complex (PDB: 6AKM and TBK1 from the AlphaFold database). TBK1-binding–defective mutants of SLMAP and SIKE1 are shown. (F and G) Gels showing that accumulation of SIKE1 and SLMAP required their interaction with TBK1. (F) HGC-27 SLMAP-KO cells or (G) SIKE1-KO cells were reconstituted with either WT or TBK1-binding–defective mutants and treated with 10 Gy IR before being harvested 2 hours after treatment. (H) Gel and plots showing that TBK1 inhibition attenuated DNA damage–induced stabilization of SIKE1 and SLMAP proteins. 293A cells pretreated with MRT (10 μM) or AA-673 (50 μM) were subjected to IR or etoposide treatment as described in F. n = 3. **P < 0.0, by 1-way ANOVA with Dunnett’s post hoc test, compared with DMSO. (I) Gel showing that inhibition of the cGAS/STING/TBK1 pathway failed to inactivate MST1/2 at an early stage after DNA damage. HGC-27 cells were treated with the indicated inhibitors and processed as in H. See also Supplemental Figure 4. ev, empty vehicle.