Fig. 7: The palmitoylation cycle regulates TLR9 ligand binding.
From: Cyclical palmitoylation regulates TLR9 signalling and systemic autoimmunity in mice
A TNF production was measured by intracellular staining in CpG B-treated Tlr9-/- RAW264.7 cells transduced with TLR9 Mut2 (n = 4 replicates). B Immunoprecipitation assay with CpG B-biotin were performed on Tlr9-/- RAW264.7 cells transduced with TLR9 WT or Mut2. A representative blot (left) and relative ligand binding (right, calculated as the ratio of CpG B-bound on cleaved mTLR9 to the total mTLR9 in input) are shown (n = 3 replicates). C TNF production was measured by ELISAs in CpG B-treated Zdhhc3+/+ or Zdhhc3-/- RAW264.7 cells transduced with mTLR9-HA (n = 3 replicates). D Immunoprecipitation assays with CpG B-biotin were performed on Zdhhc3+/+ or Zdhhc3-/- RAW264.7 cells transduced with mTLR9-HA (n = 3 replicates). E TNF production was measured by ELISAs in CpG B-treated Ppt1-/- or Ppt1-/- RAW264.7 cells transduced with mTLR9-HA (n = 4 replicates). F Immunoprecipitation assays with CpG B-biotin were performed on Ppt1+/+ or Ppt1-/- RAW264.7 cells transduced with mTLR9-HA (n = 3 replicates). G RAW264.7 cells were treated with 2-BP or HDSF overnight. TNF production was measured by intracellular staining after addition of CpG B (n = 4 replicates). H Immunoprecipitation assays with CpG B-biotin were performed on RAW264.7 cells treated with 2-BP or HDSF (n = 3 replicates). I Cell fractionation of Zdhhc3+/+ or Zdhhc3-/- RAW264.7 cells transduced with mTLR9-HA showing the distributions of TLR9 in the indicated organelle markers. A representative blot (left) and the ratio of the full length mTLR9 in Golgi (right, quantified as the ratio of boxed fractions to all fractions, n = 3 replicates). J Immunoprecipitation of TLR9-HA from the DMSO or HDSF treated RAW macrophage lines in a followed by immunoblot of UNC93B1-FLAG. UNC93B1 levels in whole-cell lysates are also shown. A representative blot (left) and the ratio of the IP UNC93B1-FLAG in HDSF treated cells to the IP UNC93B1-FLAG in DMSO treated cells, n = 3 replicates. All data are representative of three independent experiment (mean ± SEM. P values were calculated by two-way Student’s t test).
