Fig. 3: APT2 mediates the depalmitoylation of TBK1 during malarial infection.

a HEK293T cells were transfected with FLAG-EV, -PPT1, -PPT2, -APT1, or -APT2 for 24 h. TBK1 palmitoylation levels were detected by ABE assay and immunoblot analysis. This was repeated n = 3 independent times with similar results. b PEMs were treated with ML349 at the indicated concentrations for 12 h. Cell lysates were collected for ABE assay and immunoblot analysis. This was repeated n = 3 independent times with similar results. c WT or Lypla2-knockout (KO) RAW 264.7 cells were stimulated with gDNA for 12 h. Cell lysates were collected for ABE assay and immunoblot analysis. This was repeated n = 3 independent times with similar results. d HEK293T cells were transfected with FLAG-TBK1 and MYC-EV, MYC-WT APT2, or its catalytically inactivated variants (S122A and C2S) for 24 h. Cell lysates were collected for ABE assay and immunoblot analysis. This was repeated n = 3 independent times with similar results. e TBK1-KO HEK293T cells were reconstituted with plasmids encoding FLAG-WT TBK1 or FLAG-TBK1 C292S mutant and MYC-APT2 for 24 h. Cell lysates were collected for ABE assay and immunoblot analysis. This was repeated n = 3 independent times with similar results. f PEMs were stimulated with gDNA at the indicated time points. Cell lysates were collected for IP and immunoblot analysis. This was repeated n = 3 independent times with similar results. g PEMs were stimulated with gDNA, RNA, or repeated freeze-thaw cycles of N67 iRBCs for 12 h. The colocalization between TBK1 (red) and APT2 (green) was examined by confocal microscopy. The nucleus was stained with DAPI. Scale, 10 μm. The intensity analysis is next to the image. This was repeated n = 3 independent times with similar results. h PEMs were stimulated with gDNA, RNA, or repeated freeze-thaw cycles of N67 iRBCs for 12 h. The colocalization between TBK1 and APT2 was examined by PLA. The nucleus was stained with DAPI. Scale, 10 μm. Quantitative analyses of the colocalization (10 cells per group) are displayed next to the image. This was repeated n = 3 independent times with similar results. i Protein structure and molecular docking maps of TBK1 and APT2. j HEK293T cells were transfected with various combinations of plasmid encoding FLAG-WT TBK1, -R27A mutant, -C91S mutant, -V549A mutant, -Q553A mutant, -N557A mutant, or -T560R mutant of TBK1 and HA-APT2 for 24 h. Cell lysates were collected for IP (with anti-FLAG) and immunoblot analysis. This was repeated n = 3 independent times with similar results. k TBK1-KO HEK293T cells were reconstituted with plasmids encoding FLAG-WT TBK1 or FLAG-TBK1 R27A mutant and MYC-APT2 for 24 h. Cell lysates were collected for ABE assay and immunoblot analysis. This was repeated n = 3 independent times with similar results. l HEK293T cells were transfected with FLAG-TBK1 and MYC-EV, -WT APT2, -L186R APT2, or -L186K APT2 for 24 h. Cell lysates were collected for IP (with anti-FLAG) and immunoblot analysis. This was repeated n = 3 independent times with similar results. m HEK293T cells were transfected with FLAG-TBK1 and MYC-EV, -WT APT2, or -L186R APT2 for 24 h. Cell lysates were collected for ABE assay and immunoblot analysis. This was repeated n = 3 independent times with similar results. Data are presented as the mean ± SD. P-values were determined by unpaired two-tailed Student’s t tests (h). Source data are provided as a Source Data file.