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    Novus Biologicals novus ppt1
    Fig. 1 | <t>PPT1</t> deficiency protects SLE mice from autoantibodies and nephritis. C57BL/6 J (B6), Ppt1+/+ B6.Sle1yaa and Ppt1-/- B6.Sle1yaa mice were sacrificed at 16 weeks (n = 5 mice for B6; n = 15 mice for Ppt1+/+ B6.Sle1yaa and Ppt1-/- B6.Sle1yaa for the entire figure). A Spleen image (left), weight (center) and cell numbers (right) are shown. B Serum anti-DNA antibodies were measured by ELISAs. C Serum anti- RNP/Sm antibodies were measured by ELISAs at 16 weeks. D Serum total IgG were measured by ELISAs. E Glomerulus size was calculated from H&E-stained kidney sections (left). Each dot represents an individual glomerulus (n = 40 in B6; n = 44 in Ppt1+/+ B6.Sle1yaa and Ppt1-/- B6.Sle1yaa). F Representative images from kidney immunofluorescence sections are shown (left). IgG (top) and C3 (bottom) staining
    Novus Ppt1, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/novus+ppt1/PPT1+Antibody+-+Azide+and+BSA+Free/pm38169466-538-56-56
    Average 92 stars, based on 1 article reviews
    novus ppt1 - by Bioz Stars, 2026-10
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    Images

    1) Product Images from "Cyclical palmitoylation regulates TLR9 signalling and systemic autoimmunity in mice."

    Article Title: Cyclical palmitoylation regulates TLR9 signalling and systemic autoimmunity in mice.

    Journal: Nature communications

    doi: 10.1038/s41467-023-43650-z

    Fig. 1 | PPT1 deficiency protects SLE mice from autoantibodies and nephritis. C57BL/6 J (B6), Ppt1+/+ B6.Sle1yaa and Ppt1-/- B6.Sle1yaa mice were sacrificed at 16 weeks (n = 5 mice for B6; n = 15 mice for Ppt1+/+ B6.Sle1yaa and Ppt1-/- B6.Sle1yaa for the entire figure). A Spleen image (left), weight (center) and cell numbers (right) are shown. B Serum anti-DNA antibodies were measured by ELISAs. C Serum anti- RNP/Sm antibodies were measured by ELISAs at 16 weeks. D Serum total IgG were measured by ELISAs. E Glomerulus size was calculated from H&E-stained kidney sections (left). Each dot represents an individual glomerulus (n = 40 in B6; n = 44 in Ppt1+/+ B6.Sle1yaa and Ppt1-/- B6.Sle1yaa). F Representative images from kidney immunofluorescence sections are shown (left). IgG (top) and C3 (bottom) staining
    Figure Legend Snippet: Fig. 1 | PPT1 deficiency protects SLE mice from autoantibodies and nephritis. C57BL/6 J (B6), Ppt1+/+ B6.Sle1yaa and Ppt1-/- B6.Sle1yaa mice were sacrificed at 16 weeks (n = 5 mice for B6; n = 15 mice for Ppt1+/+ B6.Sle1yaa and Ppt1-/- B6.Sle1yaa for the entire figure). A Spleen image (left), weight (center) and cell numbers (right) are shown. B Serum anti-DNA antibodies were measured by ELISAs. C Serum anti- RNP/Sm antibodies were measured by ELISAs at 16 weeks. D Serum total IgG were measured by ELISAs. E Glomerulus size was calculated from H&E-stained kidney sections (left). Each dot represents an individual glomerulus (n = 40 in B6; n = 44 in Ppt1+/+ B6.Sle1yaa and Ppt1-/- B6.Sle1yaa). F Representative images from kidney immunofluorescence sections are shown (left). IgG (top) and C3 (bottom) staining

    Techniques Used: Staining

    Fig. 2 | PPT1 inhibitor HDSF suppresses IFNα in SLE patients and SLE patho- genesis in mice. A PBMCs of SLE patients were treated with DMSO or HDSF overnight. After CpG A stimulation, IFNα levels was evaluated by ELISAs (n = 17 individuals for DMSO/HDSF). B 8-weeks-old B6.Sle1yaa mice (n = 7 mice per group for the rest of figure) were treated with DMSO or HDSF for 8 weeks before sacrifice at 16 weeks. Spleen image (left), weight (center) and cell numbers (right) are shown. C Serum anti-DNA antibodies were measured by ELISAs at indicated weeks of treatment. D Serum anti-RNP/Sm antibodies were measured by ELISAs at the end of treatment. E Serum total IgG were measured by ELISAs at indicated weeks of treatment. F Glomerulus size at 16 weeks is calculated from H&E-stained kidney sections (left). Each dot represents an individual glomerulus (n = 40 glomeruli per group). G Representative images from kidney immunofluorescence sections are
    Figure Legend Snippet: Fig. 2 | PPT1 inhibitor HDSF suppresses IFNα in SLE patients and SLE patho- genesis in mice. A PBMCs of SLE patients were treated with DMSO or HDSF overnight. After CpG A stimulation, IFNα levels was evaluated by ELISAs (n = 17 individuals for DMSO/HDSF). B 8-weeks-old B6.Sle1yaa mice (n = 7 mice per group for the rest of figure) were treated with DMSO or HDSF for 8 weeks before sacrifice at 16 weeks. Spleen image (left), weight (center) and cell numbers (right) are shown. C Serum anti-DNA antibodies were measured by ELISAs at indicated weeks of treatment. D Serum anti-RNP/Sm antibodies were measured by ELISAs at the end of treatment. E Serum total IgG were measured by ELISAs at indicated weeks of treatment. F Glomerulus size at 16 weeks is calculated from H&E-stained kidney sections (left). Each dot represents an individual glomerulus (n = 40 glomeruli per group). G Representative images from kidney immunofluorescence sections are

    Techniques Used: Staining

    Fig. 6 | PPT1 depalmitoylates TLR9 in lysosomes. A ABE assays were performed on RAW264.7 cells transduced with mTLR9-HA after 4 h treatment of indicated TLR agonizts. A representative blot (left) and relative mTLR9 S-palmitoylation (right, quantified as the ratio of the mTLR9 output to the calnexin output, n = 3 replicates). B ABE assays were performed on RAW264.7 cells transduced with mTLR7-HA after 4 h treatment of R848. A representative blot (left) and relative mTLR7 S-palmitoylation (right, quantified as the ratio of the mTLR7 output to the calnexin output, n = 3 replicates). C ABE assays were performed on 293 T cells transfected with indicated depalmitoylating enzymes and mTLR9-FLAG (n = 3 replicates).
    Figure Legend Snippet: Fig. 6 | PPT1 depalmitoylates TLR9 in lysosomes. A ABE assays were performed on RAW264.7 cells transduced with mTLR9-HA after 4 h treatment of indicated TLR agonizts. A representative blot (left) and relative mTLR9 S-palmitoylation (right, quantified as the ratio of the mTLR9 output to the calnexin output, n = 3 replicates). B ABE assays were performed on RAW264.7 cells transduced with mTLR7-HA after 4 h treatment of R848. A representative blot (left) and relative mTLR7 S-palmitoylation (right, quantified as the ratio of the mTLR7 output to the calnexin output, n = 3 replicates). C ABE assays were performed on 293 T cells transfected with indicated depalmitoylating enzymes and mTLR9-FLAG (n = 3 replicates).

    Techniques Used: Transduction, Transfection

    Fig. 7 | The palmitoylation cycle regulates TLR9 ligand binding. A TNF pro- duction 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, calcu- lated 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-/-
    Figure Legend Snippet: Fig. 7 | The palmitoylation cycle regulates TLR9 ligand binding. A TNF pro- duction 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, calcu- lated 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-/-

    Techniques Used: Ligand Binding Assay, Staining, Transduction, Immunoprecipitation

    Related Articles

    Western Blot:

    Article Title: Cyclical palmitoylation regulates TLR9 signalling and systemic autoimmunity in mice
    Article Snippet: Cells were resuspended in 2 ml SHB buffer (0.25 M sucrose, 1 mM EDTA, 10 mM HEPES, pH = 7.4) plus protease inhibitor cocktail with EDTA (Roche) and 1 mM PMSF and disrupted by 30 strokes in a steel dounce homogenizer. .. The following primary antibodies used for Western blotting were all used at a dilution of 1:1000 (unless otherwise indicated): Sigma Anti-Flag (host: Mouse) Catalog #: F1840 Roche Anti-HA (host: Rat) Catalog #: 11867423001 Abcam Calnexin (host: Rabbit) Catalog #: ab213243 Abcam Rab7 (host: Rabbit) Catalog #: ab137029 Abcam Tubulin (host: Mouse) Catalog #: ab78078 (1:5000 dilution) Novus PPT1 (host: Rabbit) Catalog #: NBP2-93840 Novus TLR9 (host: Mouse) Catalog #: NBP2-24729 CST anti-biotin, HRP-linked Catalog #: 7075 S Abmart GM130 (host: Rabbit) Catalog #: T55142 CST EEA1 (host: Rabbit) Catalog #: 2411 Abcam goat anti-mouse IgG H&L (HRP) Catalog #: ab6789 (1:5000 dilution) Abcam goat anti-rabbit IgG H&L (HRP) Catalog #: ab6721 (1:5000 dilution) Abcam goat anti-rat IgG H&L (HRP) Catalog #: ab97057 (1:5000 dilution) .. Supernatants were loaded onto continuous sucrose gradients (percentage iodixanol: 0, 10, 20, 30) and ultracentrifuged in an SW41 rotor at 181,299 x g for 1 h (Optima L-90K Ultracentrifuge, Beckman Coulter).

    Article Title: Cyclical palmitoylation regulates TLR9 signalling and systemic autoimmunity in mice.
    Article Snippet: .. The following primary antibodies used for Western blotting were all used at a dilution of 1:1000 (unless otherwise indicated): Sigma Anti-Flag (host: Mouse) Catalog #: F1840 Roche Anti-HA (host: Rat) Catalog #: 11867423001 Abcam Calnexin (host: Rabbit) Catalog #: ab213243 Abcam Rab7 (host: Rabbit) Catalog #: ab137029 Abcam Tubulin (host: Mouse) Catalog #: ab78078 (1:5000 dilution) Novus PPT1 (host: Rabbit) Catalog #: NBP2-93840 Novus TLR9 (host: Mouse) Catalog #: NBP2-24729 CST anti-biotin, HRP-linked Catalog #: 7075 S Abmart GM130 (host: Rabbit) Catalog #: T55142 CST EEA1 (host: Rabbit) Catalog #: 2411 Abcamgoat anti-mouse IgGH&L (HRP) Catalog #: ab6789 (1:5000 dilution) Abcam goat anti-rabbit IgG H&L (HRP) Catalog #: ab6721 (1:5000 dilution) Abcam goat anti-rat IgG H&L (HRP) Catalog #: ab97057 (1:5000 dilution) ..



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    novus ppt1  (Novus Biologicals)
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    Novus Biologicals novus ppt1
    Fig. 1 | <t>PPT1</t> deficiency protects SLE mice from autoantibodies and nephritis. C57BL/6 J (B6), Ppt1+/+ B6.Sle1yaa and Ppt1-/- B6.Sle1yaa mice were sacrificed at 16 weeks (n = 5 mice for B6; n = 15 mice for Ppt1+/+ B6.Sle1yaa and Ppt1-/- B6.Sle1yaa for the entire figure). A Spleen image (left), weight (center) and cell numbers (right) are shown. B Serum anti-DNA antibodies were measured by ELISAs. C Serum anti- RNP/Sm antibodies were measured by ELISAs at 16 weeks. D Serum total IgG were measured by ELISAs. E Glomerulus size was calculated from H&E-stained kidney sections (left). Each dot represents an individual glomerulus (n = 40 in B6; n = 44 in Ppt1+/+ B6.Sle1yaa and Ppt1-/- B6.Sle1yaa). F Representative images from kidney immunofluorescence sections are shown (left). IgG (top) and C3 (bottom) staining
    Novus Ppt1, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/novus+ppt1/PPT1+Antibody+-+Azide+and+BSA+Free/pm38169466-538-56-56
    Average 92 stars, based on 1 article reviews
    novus ppt1 - by Bioz Stars, 2026-10
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    nbp2 93840 novus tlr9  (Novus Biologicals)
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    Novus Biologicals nbp2 93840 novus tlr9
    Fig. 4 | <t>TLR9</t> and TLR7 are S-palmitoylated. A Click chemistry reactions with azido palmitic acid (PA) were performed on RAW264.7 cells transduced with mTLR9-FLAG. A representative blot (left) and mTLR9 relative palmitoylation levels (right, quantified as the output-to-input ratio of signals from all TLR9 bands, n = 3 replicates). B ABE assays were performed in RAW264.7 cells transduced with mTLR9-FLAG (n = 3 replicates). C ABE sample preparation for mass spectrometry. D Selected MS spectra for mTLR9 C258 and C265. E ABE assays were performed on Tlr9-/- RAW264.7 cells transduced with TLR9 C258A + C265A (Mut2). mTLR9 S-palmitoylation was quantified as the mTLR9 output to calnexin output ratio (n = 4 replicates). F Click chemistry reactions with azido palmitic acid (PA) were
    Nbp2 93840 Novus Tlr9, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/novus+ppt1/PPT1+Antibody+-+Azide+and+BSA+Free/pm38169466-538-62-63
    Average 92 stars, based on 1 article reviews
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    Image Search Results


    Fig. 1 | PPT1 deficiency protects SLE mice from autoantibodies and nephritis. C57BL/6 J (B6), Ppt1+/+ B6.Sle1yaa and Ppt1-/- B6.Sle1yaa mice were sacrificed at 16 weeks (n = 5 mice for B6; n = 15 mice for Ppt1+/+ B6.Sle1yaa and Ppt1-/- B6.Sle1yaa for the entire figure). A Spleen image (left), weight (center) and cell numbers (right) are shown. B Serum anti-DNA antibodies were measured by ELISAs. C Serum anti- RNP/Sm antibodies were measured by ELISAs at 16 weeks. D Serum total IgG were measured by ELISAs. E Glomerulus size was calculated from H&E-stained kidney sections (left). Each dot represents an individual glomerulus (n = 40 in B6; n = 44 in Ppt1+/+ B6.Sle1yaa and Ppt1-/- B6.Sle1yaa). F Representative images from kidney immunofluorescence sections are shown (left). IgG (top) and C3 (bottom) staining

    Journal: Nature communications

    Article Title: Cyclical palmitoylation regulates TLR9 signalling and systemic autoimmunity in mice.

    doi: 10.1038/s41467-023-43650-z

    Figure Lengend Snippet: Fig. 1 | PPT1 deficiency protects SLE mice from autoantibodies and nephritis. C57BL/6 J (B6), Ppt1+/+ B6.Sle1yaa and Ppt1-/- B6.Sle1yaa mice were sacrificed at 16 weeks (n = 5 mice for B6; n = 15 mice for Ppt1+/+ B6.Sle1yaa and Ppt1-/- B6.Sle1yaa for the entire figure). A Spleen image (left), weight (center) and cell numbers (right) are shown. B Serum anti-DNA antibodies were measured by ELISAs. C Serum anti- RNP/Sm antibodies were measured by ELISAs at 16 weeks. D Serum total IgG were measured by ELISAs. E Glomerulus size was calculated from H&E-stained kidney sections (left). Each dot represents an individual glomerulus (n = 40 in B6; n = 44 in Ppt1+/+ B6.Sle1yaa and Ppt1-/- B6.Sle1yaa). F Representative images from kidney immunofluorescence sections are shown (left). IgG (top) and C3 (bottom) staining

    Article Snippet: The following primary antibodies used for Western blotting were all used at a dilution of 1:1000 (unless otherwise indicated): Sigma Anti-Flag (host: Mouse) Catalog #: F1840 Roche Anti-HA (host: Rat) Catalog #: 11867423001 Abcam Calnexin (host: Rabbit) Catalog #: ab213243 Abcam Rab7 (host: Rabbit) Catalog #: ab137029 Abcam Tubulin (host: Mouse) Catalog #: ab78078 (1:5000 dilution) Novus PPT1 (host: Rabbit) Catalog #: NBP2-93840 Novus TLR9 (host: Mouse) Catalog #: NBP2-24729 CST anti-biotin, HRP-linked Catalog #: 7075 S Abmart GM130 (host: Rabbit) Catalog #: T55142 CST EEA1 (host: Rabbit) Catalog #: 2411 Abcamgoat anti-mouse IgGH&L (HRP) Catalog #: ab6789 (1:5000 dilution) Abcam goat anti-rabbit IgG H&L (HRP) Catalog #: ab6721 (1:5000 dilution) Abcam goat anti-rat IgG H&L (HRP) Catalog #: ab97057 (1:5000 dilution)

    Techniques: Staining

    Fig. 2 | PPT1 inhibitor HDSF suppresses IFNα in SLE patients and SLE patho- genesis in mice. A PBMCs of SLE patients were treated with DMSO or HDSF overnight. After CpG A stimulation, IFNα levels was evaluated by ELISAs (n = 17 individuals for DMSO/HDSF). B 8-weeks-old B6.Sle1yaa mice (n = 7 mice per group for the rest of figure) were treated with DMSO or HDSF for 8 weeks before sacrifice at 16 weeks. Spleen image (left), weight (center) and cell numbers (right) are shown. C Serum anti-DNA antibodies were measured by ELISAs at indicated weeks of treatment. D Serum anti-RNP/Sm antibodies were measured by ELISAs at the end of treatment. E Serum total IgG were measured by ELISAs at indicated weeks of treatment. F Glomerulus size at 16 weeks is calculated from H&E-stained kidney sections (left). Each dot represents an individual glomerulus (n = 40 glomeruli per group). G Representative images from kidney immunofluorescence sections are

    Journal: Nature communications

    Article Title: Cyclical palmitoylation regulates TLR9 signalling and systemic autoimmunity in mice.

    doi: 10.1038/s41467-023-43650-z

    Figure Lengend Snippet: Fig. 2 | PPT1 inhibitor HDSF suppresses IFNα in SLE patients and SLE patho- genesis in mice. A PBMCs of SLE patients were treated with DMSO or HDSF overnight. After CpG A stimulation, IFNα levels was evaluated by ELISAs (n = 17 individuals for DMSO/HDSF). B 8-weeks-old B6.Sle1yaa mice (n = 7 mice per group for the rest of figure) were treated with DMSO or HDSF for 8 weeks before sacrifice at 16 weeks. Spleen image (left), weight (center) and cell numbers (right) are shown. C Serum anti-DNA antibodies were measured by ELISAs at indicated weeks of treatment. D Serum anti-RNP/Sm antibodies were measured by ELISAs at the end of treatment. E Serum total IgG were measured by ELISAs at indicated weeks of treatment. F Glomerulus size at 16 weeks is calculated from H&E-stained kidney sections (left). Each dot represents an individual glomerulus (n = 40 glomeruli per group). G Representative images from kidney immunofluorescence sections are

    Article Snippet: The following primary antibodies used for Western blotting were all used at a dilution of 1:1000 (unless otherwise indicated): Sigma Anti-Flag (host: Mouse) Catalog #: F1840 Roche Anti-HA (host: Rat) Catalog #: 11867423001 Abcam Calnexin (host: Rabbit) Catalog #: ab213243 Abcam Rab7 (host: Rabbit) Catalog #: ab137029 Abcam Tubulin (host: Mouse) Catalog #: ab78078 (1:5000 dilution) Novus PPT1 (host: Rabbit) Catalog #: NBP2-93840 Novus TLR9 (host: Mouse) Catalog #: NBP2-24729 CST anti-biotin, HRP-linked Catalog #: 7075 S Abmart GM130 (host: Rabbit) Catalog #: T55142 CST EEA1 (host: Rabbit) Catalog #: 2411 Abcamgoat anti-mouse IgGH&L (HRP) Catalog #: ab6789 (1:5000 dilution) Abcam goat anti-rabbit IgG H&L (HRP) Catalog #: ab6721 (1:5000 dilution) Abcam goat anti-rat IgG H&L (HRP) Catalog #: ab97057 (1:5000 dilution)

    Techniques: Staining

    Fig. 6 | PPT1 depalmitoylates TLR9 in lysosomes. A ABE assays were performed on RAW264.7 cells transduced with mTLR9-HA after 4 h treatment of indicated TLR agonizts. A representative blot (left) and relative mTLR9 S-palmitoylation (right, quantified as the ratio of the mTLR9 output to the calnexin output, n = 3 replicates). B ABE assays were performed on RAW264.7 cells transduced with mTLR7-HA after 4 h treatment of R848. A representative blot (left) and relative mTLR7 S-palmitoylation (right, quantified as the ratio of the mTLR7 output to the calnexin output, n = 3 replicates). C ABE assays were performed on 293 T cells transfected with indicated depalmitoylating enzymes and mTLR9-FLAG (n = 3 replicates).

    Journal: Nature communications

    Article Title: Cyclical palmitoylation regulates TLR9 signalling and systemic autoimmunity in mice.

    doi: 10.1038/s41467-023-43650-z

    Figure Lengend Snippet: Fig. 6 | PPT1 depalmitoylates TLR9 in lysosomes. A ABE assays were performed on RAW264.7 cells transduced with mTLR9-HA after 4 h treatment of indicated TLR agonizts. A representative blot (left) and relative mTLR9 S-palmitoylation (right, quantified as the ratio of the mTLR9 output to the calnexin output, n = 3 replicates). B ABE assays were performed on RAW264.7 cells transduced with mTLR7-HA after 4 h treatment of R848. A representative blot (left) and relative mTLR7 S-palmitoylation (right, quantified as the ratio of the mTLR7 output to the calnexin output, n = 3 replicates). C ABE assays were performed on 293 T cells transfected with indicated depalmitoylating enzymes and mTLR9-FLAG (n = 3 replicates).

    Article Snippet: The following primary antibodies used for Western blotting were all used at a dilution of 1:1000 (unless otherwise indicated): Sigma Anti-Flag (host: Mouse) Catalog #: F1840 Roche Anti-HA (host: Rat) Catalog #: 11867423001 Abcam Calnexin (host: Rabbit) Catalog #: ab213243 Abcam Rab7 (host: Rabbit) Catalog #: ab137029 Abcam Tubulin (host: Mouse) Catalog #: ab78078 (1:5000 dilution) Novus PPT1 (host: Rabbit) Catalog #: NBP2-93840 Novus TLR9 (host: Mouse) Catalog #: NBP2-24729 CST anti-biotin, HRP-linked Catalog #: 7075 S Abmart GM130 (host: Rabbit) Catalog #: T55142 CST EEA1 (host: Rabbit) Catalog #: 2411 Abcamgoat anti-mouse IgGH&L (HRP) Catalog #: ab6789 (1:5000 dilution) Abcam goat anti-rabbit IgG H&L (HRP) Catalog #: ab6721 (1:5000 dilution) Abcam goat anti-rat IgG H&L (HRP) Catalog #: ab97057 (1:5000 dilution)

    Techniques: Transduction, Transfection

    Fig. 7 | The palmitoylation cycle regulates TLR9 ligand binding. A TNF pro- duction 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, calcu- lated 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-/-

    Journal: Nature communications

    Article Title: Cyclical palmitoylation regulates TLR9 signalling and systemic autoimmunity in mice.

    doi: 10.1038/s41467-023-43650-z

    Figure Lengend Snippet: Fig. 7 | The palmitoylation cycle regulates TLR9 ligand binding. A TNF pro- duction 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, calcu- lated 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-/-

    Article Snippet: The following primary antibodies used for Western blotting were all used at a dilution of 1:1000 (unless otherwise indicated): Sigma Anti-Flag (host: Mouse) Catalog #: F1840 Roche Anti-HA (host: Rat) Catalog #: 11867423001 Abcam Calnexin (host: Rabbit) Catalog #: ab213243 Abcam Rab7 (host: Rabbit) Catalog #: ab137029 Abcam Tubulin (host: Mouse) Catalog #: ab78078 (1:5000 dilution) Novus PPT1 (host: Rabbit) Catalog #: NBP2-93840 Novus TLR9 (host: Mouse) Catalog #: NBP2-24729 CST anti-biotin, HRP-linked Catalog #: 7075 S Abmart GM130 (host: Rabbit) Catalog #: T55142 CST EEA1 (host: Rabbit) Catalog #: 2411 Abcamgoat anti-mouse IgGH&L (HRP) Catalog #: ab6789 (1:5000 dilution) Abcam goat anti-rabbit IgG H&L (HRP) Catalog #: ab6721 (1:5000 dilution) Abcam goat anti-rat IgG H&L (HRP) Catalog #: ab97057 (1:5000 dilution)

    Techniques: Ligand Binding Assay, Staining, Transduction, Immunoprecipitation

    Fig. 4 | TLR9 and TLR7 are S-palmitoylated. A Click chemistry reactions with azido palmitic acid (PA) were performed on RAW264.7 cells transduced with mTLR9-FLAG. A representative blot (left) and mTLR9 relative palmitoylation levels (right, quantified as the output-to-input ratio of signals from all TLR9 bands, n = 3 replicates). B ABE assays were performed in RAW264.7 cells transduced with mTLR9-FLAG (n = 3 replicates). C ABE sample preparation for mass spectrometry. D Selected MS spectra for mTLR9 C258 and C265. E ABE assays were performed on Tlr9-/- RAW264.7 cells transduced with TLR9 C258A + C265A (Mut2). mTLR9 S-palmitoylation was quantified as the mTLR9 output to calnexin output ratio (n = 4 replicates). F Click chemistry reactions with azido palmitic acid (PA) were

    Journal: Nature communications

    Article Title: Cyclical palmitoylation regulates TLR9 signalling and systemic autoimmunity in mice.

    doi: 10.1038/s41467-023-43650-z

    Figure Lengend Snippet: Fig. 4 | TLR9 and TLR7 are S-palmitoylated. A Click chemistry reactions with azido palmitic acid (PA) were performed on RAW264.7 cells transduced with mTLR9-FLAG. A representative blot (left) and mTLR9 relative palmitoylation levels (right, quantified as the output-to-input ratio of signals from all TLR9 bands, n = 3 replicates). B ABE assays were performed in RAW264.7 cells transduced with mTLR9-FLAG (n = 3 replicates). C ABE sample preparation for mass spectrometry. D Selected MS spectra for mTLR9 C258 and C265. E ABE assays were performed on Tlr9-/- RAW264.7 cells transduced with TLR9 C258A + C265A (Mut2). mTLR9 S-palmitoylation was quantified as the mTLR9 output to calnexin output ratio (n = 4 replicates). F Click chemistry reactions with azido palmitic acid (PA) were

    Article Snippet: The following primary antibodies used for Western blotting were all used at a dilution of 1:1000 (unless otherwise indicated): Sigma Anti-Flag (host: Mouse) Catalog #: F1840 Roche Anti-HA (host: Rat) Catalog #: 11867423001 Abcam Calnexin (host: Rabbit) Catalog #: ab213243 Abcam Rab7 (host: Rabbit) Catalog #: ab137029 Abcam Tubulin (host: Mouse) Catalog #: ab78078 (1:5000 dilution) Novus PPT1 (host: Rabbit) Catalog #: NBP2-93840 Novus TLR9 (host: Mouse) Catalog #: NBP2-24729 CST anti-biotin, HRP-linked Catalog #: 7075 S Abmart GM130 (host: Rabbit) Catalog #: T55142 CST EEA1 (host: Rabbit) Catalog #: 2411 Abcamgoat anti-mouse IgGH&L (HRP) Catalog #: ab6789 (1:5000 dilution) Abcam goat anti-rabbit IgG H&L (HRP) Catalog #: ab6721 (1:5000 dilution) Abcam goat anti-rat IgG H&L (HRP) Catalog #: ab97057 (1:5000 dilution)

    Techniques: Transduction, Sample Prep, Mass Spectrometry

    Fig. 5 | DHHC3 palmitoylates TLR9 in the Golgi. A ABE assays were performed in 293 T cells transfected with indicated murine DHHCs and mTLR9-FLAG. A repre- sentative blot (left) and mTLR9 relative palmitoylation levels (right, quantified as a ratio of mTLR9-FLAG signal compared to empty vector) are shown (n = 3 repli- cates). B ABE assays were performed in 293 T cells transfected with murine DHHC3 wild-type (WT) or C157S mutant, DHHC17 WT or C467S mutant, DHHC18 WT or C214S mutant, DHHC20 WT or C214S mutant, along with mTLR9-FLAG. A repre- sentative blot (left) and mTLR9 relative palmitoylation levels (right, quantified as a ratio of WT DHHC signal compared to respective mutants) are shown (n = 3 repli- cates) C TLR9 palmitoylation in DHHC3-deficient cells. ABE assays were performed on DHHC3-deficient RAW264.7 cells retrovirally transduced with mTLR9-HA. A

    Journal: Nature communications

    Article Title: Cyclical palmitoylation regulates TLR9 signalling and systemic autoimmunity in mice.

    doi: 10.1038/s41467-023-43650-z

    Figure Lengend Snippet: Fig. 5 | DHHC3 palmitoylates TLR9 in the Golgi. A ABE assays were performed in 293 T cells transfected with indicated murine DHHCs and mTLR9-FLAG. A repre- sentative blot (left) and mTLR9 relative palmitoylation levels (right, quantified as a ratio of mTLR9-FLAG signal compared to empty vector) are shown (n = 3 repli- cates). B ABE assays were performed in 293 T cells transfected with murine DHHC3 wild-type (WT) or C157S mutant, DHHC17 WT or C467S mutant, DHHC18 WT or C214S mutant, DHHC20 WT or C214S mutant, along with mTLR9-FLAG. A repre- sentative blot (left) and mTLR9 relative palmitoylation levels (right, quantified as a ratio of WT DHHC signal compared to respective mutants) are shown (n = 3 repli- cates) C TLR9 palmitoylation in DHHC3-deficient cells. ABE assays were performed on DHHC3-deficient RAW264.7 cells retrovirally transduced with mTLR9-HA. A

    Article Snippet: The following primary antibodies used for Western blotting were all used at a dilution of 1:1000 (unless otherwise indicated): Sigma Anti-Flag (host: Mouse) Catalog #: F1840 Roche Anti-HA (host: Rat) Catalog #: 11867423001 Abcam Calnexin (host: Rabbit) Catalog #: ab213243 Abcam Rab7 (host: Rabbit) Catalog #: ab137029 Abcam Tubulin (host: Mouse) Catalog #: ab78078 (1:5000 dilution) Novus PPT1 (host: Rabbit) Catalog #: NBP2-93840 Novus TLR9 (host: Mouse) Catalog #: NBP2-24729 CST anti-biotin, HRP-linked Catalog #: 7075 S Abmart GM130 (host: Rabbit) Catalog #: T55142 CST EEA1 (host: Rabbit) Catalog #: 2411 Abcamgoat anti-mouse IgGH&L (HRP) Catalog #: ab6789 (1:5000 dilution) Abcam goat anti-rabbit IgG H&L (HRP) Catalog #: ab6721 (1:5000 dilution) Abcam goat anti-rat IgG H&L (HRP) Catalog #: ab97057 (1:5000 dilution)

    Techniques: Transfection, Plasmid Preparation, Mutagenesis, Transduction

    Fig. 6 | PPT1 depalmitoylates TLR9 in lysosomes. A ABE assays were performed on RAW264.7 cells transduced with mTLR9-HA after 4 h treatment of indicated TLR agonizts. A representative blot (left) and relative mTLR9 S-palmitoylation (right, quantified as the ratio of the mTLR9 output to the calnexin output, n = 3 replicates). B ABE assays were performed on RAW264.7 cells transduced with mTLR7-HA after 4 h treatment of R848. A representative blot (left) and relative mTLR7 S-palmitoylation (right, quantified as the ratio of the mTLR7 output to the calnexin output, n = 3 replicates). C ABE assays were performed on 293 T cells transfected with indicated depalmitoylating enzymes and mTLR9-FLAG (n = 3 replicates).

    Journal: Nature communications

    Article Title: Cyclical palmitoylation regulates TLR9 signalling and systemic autoimmunity in mice.

    doi: 10.1038/s41467-023-43650-z

    Figure Lengend Snippet: Fig. 6 | PPT1 depalmitoylates TLR9 in lysosomes. A ABE assays were performed on RAW264.7 cells transduced with mTLR9-HA after 4 h treatment of indicated TLR agonizts. A representative blot (left) and relative mTLR9 S-palmitoylation (right, quantified as the ratio of the mTLR9 output to the calnexin output, n = 3 replicates). B ABE assays were performed on RAW264.7 cells transduced with mTLR7-HA after 4 h treatment of R848. A representative blot (left) and relative mTLR7 S-palmitoylation (right, quantified as the ratio of the mTLR7 output to the calnexin output, n = 3 replicates). C ABE assays were performed on 293 T cells transfected with indicated depalmitoylating enzymes and mTLR9-FLAG (n = 3 replicates).

    Article Snippet: The following primary antibodies used for Western blotting were all used at a dilution of 1:1000 (unless otherwise indicated): Sigma Anti-Flag (host: Mouse) Catalog #: F1840 Roche Anti-HA (host: Rat) Catalog #: 11867423001 Abcam Calnexin (host: Rabbit) Catalog #: ab213243 Abcam Rab7 (host: Rabbit) Catalog #: ab137029 Abcam Tubulin (host: Mouse) Catalog #: ab78078 (1:5000 dilution) Novus PPT1 (host: Rabbit) Catalog #: NBP2-93840 Novus TLR9 (host: Mouse) Catalog #: NBP2-24729 CST anti-biotin, HRP-linked Catalog #: 7075 S Abmart GM130 (host: Rabbit) Catalog #: T55142 CST EEA1 (host: Rabbit) Catalog #: 2411 Abcamgoat anti-mouse IgGH&L (HRP) Catalog #: ab6789 (1:5000 dilution) Abcam goat anti-rabbit IgG H&L (HRP) Catalog #: ab6721 (1:5000 dilution) Abcam goat anti-rat IgG H&L (HRP) Catalog #: ab97057 (1:5000 dilution)

    Techniques: Transduction, Transfection

    Fig. 7 | The palmitoylation cycle regulates TLR9 ligand binding. A TNF pro- duction 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, calcu- lated 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-/-

    Journal: Nature communications

    Article Title: Cyclical palmitoylation regulates TLR9 signalling and systemic autoimmunity in mice.

    doi: 10.1038/s41467-023-43650-z

    Figure Lengend Snippet: Fig. 7 | The palmitoylation cycle regulates TLR9 ligand binding. A TNF pro- duction 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, calcu- lated 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-/-

    Article Snippet: The following primary antibodies used for Western blotting were all used at a dilution of 1:1000 (unless otherwise indicated): Sigma Anti-Flag (host: Mouse) Catalog #: F1840 Roche Anti-HA (host: Rat) Catalog #: 11867423001 Abcam Calnexin (host: Rabbit) Catalog #: ab213243 Abcam Rab7 (host: Rabbit) Catalog #: ab137029 Abcam Tubulin (host: Mouse) Catalog #: ab78078 (1:5000 dilution) Novus PPT1 (host: Rabbit) Catalog #: NBP2-93840 Novus TLR9 (host: Mouse) Catalog #: NBP2-24729 CST anti-biotin, HRP-linked Catalog #: 7075 S Abmart GM130 (host: Rabbit) Catalog #: T55142 CST EEA1 (host: Rabbit) Catalog #: 2411 Abcamgoat anti-mouse IgGH&L (HRP) Catalog #: ab6789 (1:5000 dilution) Abcam goat anti-rabbit IgG H&L (HRP) Catalog #: ab6721 (1:5000 dilution) Abcam goat anti-rat IgG H&L (HRP) Catalog #: ab97057 (1:5000 dilution)

    Techniques: Ligand Binding Assay, Staining, Transduction, Immunoprecipitation