Fig. 2: ZDHHC9 mediates TBK1 palmitoylation.

a Cell lysates from experiments in Fig. 1 (h) were subjected to acyl-biotin exchange (ABE) assays and immunoblot analysis to assess TBK1 palmitoylation. This was repeated n = 3 independent times with similar results. b Human embryonic kidney (HEK) 293 T cells were transfected with Flag-TBK1 for 24 h, followed by the treatment of DMSO (vehicle) or 2-BP (100 mM for 12 h). TBK1 palmitoylation and protein levels were assessed by ABE assay and immunoblot analysis. This was repeated n = 3 independent times with similar results. c BMDMs were stimulated with gDNA at the indicated time points, and TBK1 palmitoylation levels were measured using ABE assay and immunoblotting. This was repeated n = 3 independent times with similar results. d HEK293T cells were transfected with wild-type (WT) or deletion mutants of Flag-TBK1 for 24 h, and TBK1 palmitoylation levels were measured using ABE assay and immunoblotting. This was repeated n = 3 independent times with similar results. e Comparative analysis of potential palmitoylation sites within TBK1 sequences across different species. f HEK293T cells were transfected with WT FLAG-TBK1 or TBK1 palmitoylation-deficient mutants for 24 h. TBK1 palmitoylation levels were detected by ABE assay and immunoblot analysis. This was repeated n = 3 independent times with similar results. g Heatmap analysis of expression changes in the ZDHHC gene family in PEMs, with or without stimulation following freeze-thawed iRBCs. Each group has three biological replicates. h The relative mRNA levels of Zdhhc genes in PEMs were determined by RT-qPCR following N67 infection, with mRNA levels was normalized to Gapdh (n = 3 biological replicates). i RAW 264.7 cells were transfected with Scr shRNA or Zdhhc shRNAs for 24 h, followed by transfection with FLAG-TBK1. TBK1 palmitoylation and protein levels were measured using the ABE assay and immunoblotting. This was repeated n = 3 independent times with similar results. j, k HEK293T cells were transfected with either WT HA-ZDHHC9 or the enzymatically inactive mutant ZDHHC9 (C169S) for 24 h (j). TBK1-KO HEK293T cells were reconstituted with plasmids encoding WT FLAG-TBK1 or FLAG-TBK1 C292S mutant for 24 h (k). Cell lysates were harvested for ABE assay and immunoblot analysis. This was repeated n = 3 independent times with similar results. l Proximity ligation assay (PLA) was performed on HEK293T cells overexpressing FLAG-ZDHHC9 to examine the colocalization of TBK1 and FLAG. The nucleus was stained with DAPI. Scale, 10 μm. Quantitative analyses of the colocalization (10 cells per group) are presented next to the image. This was repeated n = 3 independent times with similar results. m PEMs were stimulated with freeze-thawed iRBCs for 12 h. The colocalization between TBK1 (red) and ZDHHC9 (green) was examined by confocal microscopy. The nucleus was stained with DAPI (blue). Scale, 10 μm. The intensity analysis is next to the image. This was repeated n = 3 independent times with similar results. n, o RAW 264.7 cells were transfected with Scr shRNA or Zdhhc9 shRNA for 24 h, followed by stimulation with gDNA at the indicated time points. Cell lysates were used for RT-qPCR analysis (n = 3 biological replicates) (n). IFN-β release in the supernatants was determined by ELISA (n = 3 biological replicates) (o). p, q RAW 264.7 cells were transfected with HA-EV, WT HA-ZDHHC9, or the enzymatically inactive mutant of ZDHHC9 (C169S) for 24 h, and then stimulated with gDNA at the indicated time points. Cell lysates were collected for immunoblotting. This was repeated n = 3 independent times with similar results (p) and RT-qPCR analysis (n = 3 biological replicates) (q). Data are presented as the mean ± SD. P-values were determined by unpaired two tailed Student’s t tests (l, n, o, q). Source data are provided as a Source Data file.