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Proteintech tlr4
Tlr4, supplied by Proteintech, used in various techniques. Bioz Stars score: 96/100, based on 1338 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/tlr4/TLR4+Antibody/pm42897266-297-11-12
Average 96 stars, based on 1338 article reviews
tlr4 - by Bioz Stars, 2026-11
96/100 stars

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Article Title: A novel hydrogen persulfide donor HPD1 alleviated LPS-induced neuroinflammation by modulating glial polarization through TLR4-MAPK/NF-κB pathway
Article Snippet: Primary , TLR4 , Mouse , Mouse , 1:4000 , Proteintech (66350-1-Ig).

Cell-Signaling:

Article Title: Baicalin attenuates myocardial ischemia-reperfusion injury by modulating TLR4/MD-2-associated inflammatory responses and reducing neutrophil‑related tissue injury
Article Snippet: .. The following primary antibodies were used: TLR4 (1:1000, 19811-1-AP, Proteintech), p-NF-κB p65 (Ser536) (1:1000, 3033P, Cell Signaling), NLRP3 (1:1000, ET1610-93, HuaBio), IL-1β (1:1000, HA601036, HuaBio), and β-actin (1:1000, HA722023, HuaBio). .. The next day, after washing with TBST, the membranes were incubated with HRP-conjugated goat anti-rabbit or anti-mouse IgG secondary antibodies at room temperature for 2 h. Protein bands were visualized using ECL chemiluminescence reagent, imaged with a chemiluminescence imaging system, and analyzed by densitometry using ImageJ software (version 1.53t; https://imagej.nih.gov/ij/).

Article Title: Pterostilbene attenuates cisplatin-induced acute kidney injury by suppressing TLR4/MyD88/NF-κB signaling.
Article Snippet: Immunoreactive bands were visualized using an enhanced chemiluminescence (ECL; Biosharp, China) detection system, and band intensities were quantified using ImageJ software. .. The main details of the antibodies were as follows: KIM-1 (1:800, Abcam, USA); TNF-α (1:1000, Proteintech Group Inc., China); IL-1β (1:1000, Proteintech Group Inc., China); IL-6 (1:1000, Proteintech Group Inc., China); COX-2 (1:1000, Proteintech Group Inc., China); iNOS (1:1000, Proteintech Group Inc., China); Bax (1:1000, Abcam, USA); Bcl-2 (1:3000, Proteintech Group Inc., China); Caspase-3 (1:1000, Cell Signaling Technology, USA); Cleaved Caspase-3 (1:1000, Cell Signaling Technology, USA); TLR4 (1:1000, Proteintech Group Inc., China); MyD88 (1:1000, Proteintech Group Inc., China); NF-κB p65 (1:1000, Proteintech Group Inc., China); p-p65 (1:1000, Proteintech Group Inc., China); GAPDH (1:50000, Proteintech Group Inc., China); β-actin (1:20000, Proteintech Group Inc., China); HRP-conjugated goat Anti-Rabbit/Anti-Mouse IgG (H+L) (1:10000, Proteintech Group Inc., China). ..

Article Title: Monocyte surface Y RNAs potentiate innate immunity.
Article Snippet: RNAs can associate with the surface of diverse cell types, yet the functions of specific RNA species on primary immune cells remain unclear.. Here, utilizing methods developed to profile, image and quantify surface RNAs in rare primary cell populations, we found that Y RNAs, a family of small noncoding RNAs, preferentially localize to human monocyte surface.. In an exploratory pooled inflammatory bowel disease cohort, the proportion of cell-surface-Y-RNA-positive peripheral monocytes was markedly higher in patients with inflammatory bowel disease than in healthy donors or individuals with irritable bowel syndrome.

Incubation:

Article Title: Ruscogenin attenuates sepsis-induced multiple organ injury by inhibiting NMMHC IIA.
Article Snippet: .. The membranes were blocked with 5% skimmed milk and incubated with primary antibodies against NMMHC IIA (Proteintech, CAT#11128-1-AP, RRID: AB_2147294), E-selectin (Affinity Biosciences, Cat# DF6914, RRID: AB_2838873), ICAM-1 (Proteintech, CAT#60299-1-IG, RRID: AB_2881414), VE-cadherin (Affinity Biosciences, Cat# AF6265, RRID: AB_2835123), TLR4 (Proteintech, CAT19811-1-AP, RRID: AB_10638446), MyD88 (Proteintech, CAT#67969-1-Ig, RRID: AB_2918720), NF-κB (Proteintech, CAT#80979-1-RR, RRID: AB_2918923), pNF-κB (Affinity Biosciences, CAT#AF2006, RRID: AB_2834435), Beta Actin (β-actin, Proteintech, CAT#66009-1-Ig, RRID: AB_2687938) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Proteintech, CAT#60004-1-Ig, RRID:AB_2107436) overnight at 4 ◦C. .. Subsequently, the membranes were incubated with secondary anti-rabbit (Bioworld, CAT#BS20241-Y) or anti-mouse (Bioworld, CAT#BS20242-Y) IgG conjugated with horseradish peroxidase (15,000 dilution).

Article Title: Combined metabolomics and network pharmacology explore the protective mechanism of bornyl acetate against cognitive impairment in chronic sleep-deprived mice
Article Snippet: .. The membranes were blocked by immersion in a solution of 5% BSA, and were incubated at 4 °C overnight with the following primary antibodies: Iba-1 (1:1500, 10904-1-AP, Proteintech), TLR4 (1:4500, 66350-1-lg, Proteintech), IRAK1 (1:2000, 10478-2-AP, Proteintech), TRAF6 (1:2000, AF5376, Affinity), NF-κB p65 (1:5000, 10745-1-AP, Proteintech), p-NF-κB p65 (1:5000, Affinity, AF2006), Beta Actin (1:3000, 20536- 1-AP, Proteintech). .. The next day the membranes were placed in HRP-linked secondary antibodies (1:6000, SA00001-1, Proteintech; 1:6000, SA00001-2, Proteintech) incubated for 2 h at room temperature.

Article Title: Inflammatory landscape of Gardnerella vaginalis -associated bacterial vaginosis in rats: an integrated multi-omics analysis
Article Snippet: .. After blocking with a rapid blocking buffer (Epizyme, Shanghai, China), the blots were incubated overnight at 4 °C with the following primary antibodies: TLR2 (Abcam, Cambridge, UK; dilution 1:1000), TLR4 (Proteintech, Hubei, China; dilution 1:1000), phospho-NF-κB p65 at serine 536 (pS536-p65; Proteintech; dilution 1:2000), cyclooxygenase-2 (COX-2; Proteintech; dilution 1:1000), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Proteintech; dilution 1:10000). .. The next day, the membranes were incubated with HRP-conjugated secondary antibodies (Proteintech; dilution 1:10000) for 1 h at room temperature ( ).

Chloramphenicol Acetyltransferase Assay:

Article Title: Ruscogenin attenuates sepsis-induced multiple organ injury by inhibiting NMMHC IIA.
Article Snippet: .. The membranes were blocked with 5% skimmed milk and incubated with primary antibodies against NMMHC IIA (Proteintech, CAT#11128-1-AP, RRID: AB_2147294), E-selectin (Affinity Biosciences, Cat# DF6914, RRID: AB_2838873), ICAM-1 (Proteintech, CAT#60299-1-IG, RRID: AB_2881414), VE-cadherin (Affinity Biosciences, Cat# AF6265, RRID: AB_2835123), TLR4 (Proteintech, CAT19811-1-AP, RRID: AB_10638446), MyD88 (Proteintech, CAT#67969-1-Ig, RRID: AB_2918720), NF-κB (Proteintech, CAT#80979-1-RR, RRID: AB_2918923), pNF-κB (Affinity Biosciences, CAT#AF2006, RRID: AB_2834435), Beta Actin (β-actin, Proteintech, CAT#66009-1-Ig, RRID: AB_2687938) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Proteintech, CAT#60004-1-Ig, RRID:AB_2107436) overnight at 4 ◦C. .. Subsequently, the membranes were incubated with secondary anti-rabbit (Bioworld, CAT#BS20241-Y) or anti-mouse (Bioworld, CAT#BS20242-Y) IgG conjugated with horseradish peroxidase (15,000 dilution).

Blocking Assay:

Article Title: Inflammatory landscape of Gardnerella vaginalis -associated bacterial vaginosis in rats: an integrated multi-omics analysis
Article Snippet: .. After blocking with a rapid blocking buffer (Epizyme, Shanghai, China), the blots were incubated overnight at 4 °C with the following primary antibodies: TLR2 (Abcam, Cambridge, UK; dilution 1:1000), TLR4 (Proteintech, Hubei, China; dilution 1:1000), phospho-NF-κB p65 at serine 536 (pS536-p65; Proteintech; dilution 1:2000), cyclooxygenase-2 (COX-2; Proteintech; dilution 1:1000), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Proteintech; dilution 1:10000). .. The next day, the membranes were incubated with HRP-conjugated secondary antibodies (Proteintech; dilution 1:10000) for 1 h at room temperature ( ).



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BMAd‐derived sDPP4 activates BMM STING signaling through TLR4 binding and mtDNA cooperation. (a) Schematic diagram of sDPP4‐mediated STING activation. (b) Representative immunoblots of TLR2 and TLR4 protein levels in BMMs from rDPP4‐treated or SN‐rDPP4‐treated male mice ( n = 3 biological replicates). (c) Representative immunoblots of TAK1 and STING protein/phosphorylation levels in BMMs stimulated with SN‐rDPP4 and treated with C29 or Resatorvid ( n = 3 biological replicates). (d) Molecular docking analysis of the direct binding mode between the TLR4/MD‐2 complex and the DPP4 structure. (e) SPR analysis of the direct interaction between rDPP4 and <t>rTLR4.</t> (f) Co‐Immunoprecipitation analysis of Flag‐ Dpp4 and HA‐ Tlr4 binding. (g) Co‐Immunoprecipitation analysis of DPP4 deletion mutants binding to full‐length TLR4. (h) qPCR analysis of cytosolic DNA extracted from the cytosolic extracts of 3 M‐/24 M‐BMMs ( n = 6 biological replicates). (i,j) Representative immunofluorescence staining of s anti‐dsDNA (green), anti‐Hsp60 (red), and DAPI (blue) in 3 M‐/18 M BMMs (scale bar, 1 µm) (i). The percentage of dsDNA (green) outside of mitochondria (red) was quantified (j) ( n = 15 from 3 biological replicates). (k) Representative immunoblots of cGAS, STING, TBK1, IRF3, NF‐κB, and IκB protein/phosphorylation levels in BMMs treated with EtBr ( n = 3 biological replicates). (l) Flow cytometry analysis and quantification of mtROS production in WT‐3 M‐/WT‐18 M‐BMMs and Sting ΔMφ ‐18 M‐BMMs ( n = 6 biological replicates). (m) Flow cytometry analysis and quantification of mtROS production in SN‐rDPP4‐treated or co‐cultured BMMs with or without Cgas and Sting knockdown ( n = 6 biological replicates). Data are shown as mean ± SD.
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BMAd‐derived sDPP4 activates BMM STING signaling through TLR4 binding and mtDNA cooperation. (a) Schematic diagram of sDPP4‐mediated STING activation. (b) Representative immunoblots of TLR2 and TLR4 protein levels in BMMs from rDPP4‐treated or SN‐rDPP4‐treated male mice ( n = 3 biological replicates). (c) Representative immunoblots of TAK1 and STING protein/phosphorylation levels in BMMs stimulated with SN‐rDPP4 and treated with C29 or Resatorvid ( n = 3 biological replicates). (d) Molecular docking analysis of the direct binding mode between the TLR4/MD‐2 complex and the DPP4 structure. (e) SPR analysis of the direct interaction between rDPP4 and <t>rTLR4.</t> (f) Co‐Immunoprecipitation analysis of Flag‐ Dpp4 and HA‐ Tlr4 binding. (g) Co‐Immunoprecipitation analysis of DPP4 deletion mutants binding to full‐length TLR4. (h) qPCR analysis of cytosolic DNA extracted from the cytosolic extracts of 3 M‐/24 M‐BMMs ( n = 6 biological replicates). (i,j) Representative immunofluorescence staining of s anti‐dsDNA (green), anti‐Hsp60 (red), and DAPI (blue) in 3 M‐/18 M BMMs (scale bar, 1 µm) (i). The percentage of dsDNA (green) outside of mitochondria (red) was quantified (j) ( n = 15 from 3 biological replicates). (k) Representative immunoblots of cGAS, STING, TBK1, IRF3, NF‐κB, and IκB protein/phosphorylation levels in BMMs treated with EtBr ( n = 3 biological replicates). (l) Flow cytometry analysis and quantification of mtROS production in WT‐3 M‐/WT‐18 M‐BMMs and Sting ΔMφ ‐18 M‐BMMs ( n = 6 biological replicates). (m) Flow cytometry analysis and quantification of mtROS production in SN‐rDPP4‐treated or co‐cultured BMMs with or without Cgas and Sting knockdown ( n = 6 biological replicates). Data are shown as mean ± SD.
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BMAd‐derived sDPP4 activates BMM STING signaling through TLR4 binding and mtDNA cooperation. (a) Schematic diagram of sDPP4‐mediated STING activation. (b) Representative immunoblots of TLR2 and TLR4 protein levels in BMMs from rDPP4‐treated or SN‐rDPP4‐treated male mice ( n = 3 biological replicates). (c) Representative immunoblots of TAK1 and STING protein/phosphorylation levels in BMMs stimulated with SN‐rDPP4 and treated with C29 or Resatorvid ( n = 3 biological replicates). (d) Molecular docking analysis of the direct binding mode between the TLR4/MD‐2 complex and the DPP4 structure. (e) SPR analysis of the direct interaction between rDPP4 and <t>rTLR4.</t> (f) Co‐Immunoprecipitation analysis of Flag‐ Dpp4 and HA‐ Tlr4 binding. (g) Co‐Immunoprecipitation analysis of DPP4 deletion mutants binding to full‐length TLR4. (h) qPCR analysis of cytosolic DNA extracted from the cytosolic extracts of 3 M‐/24 M‐BMMs ( n = 6 biological replicates). (i,j) Representative immunofluorescence staining of s anti‐dsDNA (green), anti‐Hsp60 (red), and DAPI (blue) in 3 M‐/18 M BMMs (scale bar, 1 µm) (i). The percentage of dsDNA (green) outside of mitochondria (red) was quantified (j) ( n = 15 from 3 biological replicates). (k) Representative immunoblots of cGAS, STING, TBK1, IRF3, NF‐κB, and IκB protein/phosphorylation levels in BMMs treated with EtBr ( n = 3 biological replicates). (l) Flow cytometry analysis and quantification of mtROS production in WT‐3 M‐/WT‐18 M‐BMMs and Sting ΔMφ ‐18 M‐BMMs ( n = 6 biological replicates). (m) Flow cytometry analysis and quantification of mtROS production in SN‐rDPP4‐treated or co‐cultured BMMs with or without Cgas and Sting knockdown ( n = 6 biological replicates). Data are shown as mean ± SD.
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BMAd‐derived sDPP4 activates BMM STING signaling through TLR4 binding and mtDNA cooperation. (a) Schematic diagram of sDPP4‐mediated STING activation. (b) Representative immunoblots of TLR2 and TLR4 protein levels in BMMs from rDPP4‐treated or SN‐rDPP4‐treated male mice ( n = 3 biological replicates). (c) Representative immunoblots of TAK1 and STING protein/phosphorylation levels in BMMs stimulated with SN‐rDPP4 and treated with C29 or Resatorvid ( n = 3 biological replicates). (d) Molecular docking analysis of the direct binding mode between the TLR4/MD‐2 complex and the DPP4 structure. (e) SPR analysis of the direct interaction between rDPP4 and <t>rTLR4.</t> (f) Co‐Immunoprecipitation analysis of Flag‐ Dpp4 and HA‐ Tlr4 binding. (g) Co‐Immunoprecipitation analysis of DPP4 deletion mutants binding to full‐length TLR4. (h) qPCR analysis of cytosolic DNA extracted from the cytosolic extracts of 3 M‐/24 M‐BMMs ( n = 6 biological replicates). (i,j) Representative immunofluorescence staining of s anti‐dsDNA (green), anti‐Hsp60 (red), and DAPI (blue) in 3 M‐/18 M BMMs (scale bar, 1 µm) (i). The percentage of dsDNA (green) outside of mitochondria (red) was quantified (j) ( n = 15 from 3 biological replicates). (k) Representative immunoblots of cGAS, STING, TBK1, IRF3, NF‐κB, and IκB protein/phosphorylation levels in BMMs treated with EtBr ( n = 3 biological replicates). (l) Flow cytometry analysis and quantification of mtROS production in WT‐3 M‐/WT‐18 M‐BMMs and Sting ΔMφ ‐18 M‐BMMs ( n = 6 biological replicates). (m) Flow cytometry analysis and quantification of mtROS production in SN‐rDPP4‐treated or co‐cultured BMMs with or without Cgas and Sting knockdown ( n = 6 biological replicates). Data are shown as mean ± SD.
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BMAd‐derived sDPP4 activates BMM STING signaling through TLR4 binding and mtDNA cooperation. (a) Schematic diagram of sDPP4‐mediated STING activation. (b) Representative immunoblots of TLR2 and TLR4 protein levels in BMMs from rDPP4‐treated or SN‐rDPP4‐treated male mice ( n = 3 biological replicates). (c) Representative immunoblots of TAK1 and STING protein/phosphorylation levels in BMMs stimulated with SN‐rDPP4 and treated with C29 or Resatorvid ( n = 3 biological replicates). (d) Molecular docking analysis of the direct binding mode between the TLR4/MD‐2 complex and the DPP4 structure. (e) SPR analysis of the direct interaction between rDPP4 and <t>rTLR4.</t> (f) Co‐Immunoprecipitation analysis of Flag‐ Dpp4 and HA‐ Tlr4 binding. (g) Co‐Immunoprecipitation analysis of DPP4 deletion mutants binding to full‐length TLR4. (h) qPCR analysis of cytosolic DNA extracted from the cytosolic extracts of 3 M‐/24 M‐BMMs ( n = 6 biological replicates). (i,j) Representative immunofluorescence staining of s anti‐dsDNA (green), anti‐Hsp60 (red), and DAPI (blue) in 3 M‐/18 M BMMs (scale bar, 1 µm) (i). The percentage of dsDNA (green) outside of mitochondria (red) was quantified (j) ( n = 15 from 3 biological replicates). (k) Representative immunoblots of cGAS, STING, TBK1, IRF3, NF‐κB, and IκB protein/phosphorylation levels in BMMs treated with EtBr ( n = 3 biological replicates). (l) Flow cytometry analysis and quantification of mtROS production in WT‐3 M‐/WT‐18 M‐BMMs and Sting ΔMφ ‐18 M‐BMMs ( n = 6 biological replicates). (m) Flow cytometry analysis and quantification of mtROS production in SN‐rDPP4‐treated or co‐cultured BMMs with or without Cgas and Sting knockdown ( n = 6 biological replicates). Data are shown as mean ± SD.
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BMAd‐derived sDPP4 activates BMM STING signaling through TLR4 binding and mtDNA cooperation. (a) Schematic diagram of sDPP4‐mediated STING activation. (b) Representative immunoblots of TLR2 and TLR4 protein levels in BMMs from rDPP4‐treated or SN‐rDPP4‐treated male mice ( n = 3 biological replicates). (c) Representative immunoblots of TAK1 and STING protein/phosphorylation levels in BMMs stimulated with SN‐rDPP4 and treated with C29 or Resatorvid ( n = 3 biological replicates). (d) Molecular docking analysis of the direct binding mode between the TLR4/MD‐2 complex and the DPP4 structure. (e) SPR analysis of the direct interaction between rDPP4 and <t>rTLR4.</t> (f) Co‐Immunoprecipitation analysis of Flag‐ Dpp4 and HA‐ Tlr4 binding. (g) Co‐Immunoprecipitation analysis of DPP4 deletion mutants binding to full‐length TLR4. (h) qPCR analysis of cytosolic DNA extracted from the cytosolic extracts of 3 M‐/24 M‐BMMs ( n = 6 biological replicates). (i,j) Representative immunofluorescence staining of s anti‐dsDNA (green), anti‐Hsp60 (red), and DAPI (blue) in 3 M‐/18 M BMMs (scale bar, 1 µm) (i). The percentage of dsDNA (green) outside of mitochondria (red) was quantified (j) ( n = 15 from 3 biological replicates). (k) Representative immunoblots of cGAS, STING, TBK1, IRF3, NF‐κB, and IκB protein/phosphorylation levels in BMMs treated with EtBr ( n = 3 biological replicates). (l) Flow cytometry analysis and quantification of mtROS production in WT‐3 M‐/WT‐18 M‐BMMs and Sting ΔMφ ‐18 M‐BMMs ( n = 6 biological replicates). (m) Flow cytometry analysis and quantification of mtROS production in SN‐rDPP4‐treated or co‐cultured BMMs with or without Cgas and Sting knockdown ( n = 6 biological replicates). Data are shown as mean ± SD.
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94
Cyagen Biosciences tlr4fl
BMAd‐derived sDPP4 activates BMM STING signaling through TLR4 binding and mtDNA cooperation. (a) Schematic diagram of sDPP4‐mediated STING activation. (b) Representative immunoblots of TLR2 and TLR4 protein levels in BMMs from rDPP4‐treated or SN‐rDPP4‐treated male mice ( n = 3 biological replicates). (c) Representative immunoblots of TAK1 and STING protein/phosphorylation levels in BMMs stimulated with SN‐rDPP4 and treated with C29 or Resatorvid ( n = 3 biological replicates). (d) Molecular docking analysis of the direct binding mode between the TLR4/MD‐2 complex and the DPP4 structure. (e) SPR analysis of the direct interaction between rDPP4 and <t>rTLR4.</t> (f) Co‐Immunoprecipitation analysis of Flag‐ Dpp4 and HA‐ Tlr4 binding. (g) Co‐Immunoprecipitation analysis of DPP4 deletion mutants binding to full‐length TLR4. (h) qPCR analysis of cytosolic DNA extracted from the cytosolic extracts of 3 M‐/24 M‐BMMs ( n = 6 biological replicates). (i,j) Representative immunofluorescence staining of s anti‐dsDNA (green), anti‐Hsp60 (red), and DAPI (blue) in 3 M‐/18 M BMMs (scale bar, 1 µm) (i). The percentage of dsDNA (green) outside of mitochondria (red) was quantified (j) ( n = 15 from 3 biological replicates). (k) Representative immunoblots of cGAS, STING, TBK1, IRF3, NF‐κB, and IκB protein/phosphorylation levels in BMMs treated with EtBr ( n = 3 biological replicates). (l) Flow cytometry analysis and quantification of mtROS production in WT‐3 M‐/WT‐18 M‐BMMs and Sting ΔMφ ‐18 M‐BMMs ( n = 6 biological replicates). (m) Flow cytometry analysis and quantification of mtROS production in SN‐rDPP4‐treated or co‐cultured BMMs with or without Cgas and Sting knockdown ( n = 6 biological replicates). Data are shown as mean ± SD.
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BMAd‐derived sDPP4 activates BMM STING signaling through TLR4 binding and mtDNA cooperation. (a) Schematic diagram of sDPP4‐mediated STING activation. (b) Representative immunoblots of TLR2 and TLR4 protein levels in BMMs from rDPP4‐treated or SN‐rDPP4‐treated male mice ( n = 3 biological replicates). (c) Representative immunoblots of TAK1 and STING protein/phosphorylation levels in BMMs stimulated with SN‐rDPP4 and treated with C29 or Resatorvid ( n = 3 biological replicates). (d) Molecular docking analysis of the direct binding mode between the TLR4/MD‐2 complex and the DPP4 structure. (e) SPR analysis of the direct interaction between rDPP4 and rTLR4. (f) Co‐Immunoprecipitation analysis of Flag‐ Dpp4 and HA‐ Tlr4 binding. (g) Co‐Immunoprecipitation analysis of DPP4 deletion mutants binding to full‐length TLR4. (h) qPCR analysis of cytosolic DNA extracted from the cytosolic extracts of 3 M‐/24 M‐BMMs ( n = 6 biological replicates). (i,j) Representative immunofluorescence staining of s anti‐dsDNA (green), anti‐Hsp60 (red), and DAPI (blue) in 3 M‐/18 M BMMs (scale bar, 1 µm) (i). The percentage of dsDNA (green) outside of mitochondria (red) was quantified (j) ( n = 15 from 3 biological replicates). (k) Representative immunoblots of cGAS, STING, TBK1, IRF3, NF‐κB, and IκB protein/phosphorylation levels in BMMs treated with EtBr ( n = 3 biological replicates). (l) Flow cytometry analysis and quantification of mtROS production in WT‐3 M‐/WT‐18 M‐BMMs and Sting ΔMφ ‐18 M‐BMMs ( n = 6 biological replicates). (m) Flow cytometry analysis and quantification of mtROS production in SN‐rDPP4‐treated or co‐cultured BMMs with or without Cgas and Sting knockdown ( n = 6 biological replicates). Data are shown as mean ± SD.

Journal: Advanced Science

Article Title: Bone Marrow Macrophage STING Signaling is a Direct Nexus Linking Marrow Adipose Expansion to NAD + Depletion and Osteogenic Decline in Skeletal Aging

doi: 10.1002/advs.77571

Figure Lengend Snippet: BMAd‐derived sDPP4 activates BMM STING signaling through TLR4 binding and mtDNA cooperation. (a) Schematic diagram of sDPP4‐mediated STING activation. (b) Representative immunoblots of TLR2 and TLR4 protein levels in BMMs from rDPP4‐treated or SN‐rDPP4‐treated male mice ( n = 3 biological replicates). (c) Representative immunoblots of TAK1 and STING protein/phosphorylation levels in BMMs stimulated with SN‐rDPP4 and treated with C29 or Resatorvid ( n = 3 biological replicates). (d) Molecular docking analysis of the direct binding mode between the TLR4/MD‐2 complex and the DPP4 structure. (e) SPR analysis of the direct interaction between rDPP4 and rTLR4. (f) Co‐Immunoprecipitation analysis of Flag‐ Dpp4 and HA‐ Tlr4 binding. (g) Co‐Immunoprecipitation analysis of DPP4 deletion mutants binding to full‐length TLR4. (h) qPCR analysis of cytosolic DNA extracted from the cytosolic extracts of 3 M‐/24 M‐BMMs ( n = 6 biological replicates). (i,j) Representative immunofluorescence staining of s anti‐dsDNA (green), anti‐Hsp60 (red), and DAPI (blue) in 3 M‐/18 M BMMs (scale bar, 1 µm) (i). The percentage of dsDNA (green) outside of mitochondria (red) was quantified (j) ( n = 15 from 3 biological replicates). (k) Representative immunoblots of cGAS, STING, TBK1, IRF3, NF‐κB, and IκB protein/phosphorylation levels in BMMs treated with EtBr ( n = 3 biological replicates). (l) Flow cytometry analysis and quantification of mtROS production in WT‐3 M‐/WT‐18 M‐BMMs and Sting ΔMφ ‐18 M‐BMMs ( n = 6 biological replicates). (m) Flow cytometry analysis and quantification of mtROS production in SN‐rDPP4‐treated or co‐cultured BMMs with or without Cgas and Sting knockdown ( n = 6 biological replicates). Data are shown as mean ± SD.

Article Snippet: The binding interaction between rTLR4 (Sino Biological, 10146‐H08B) and rDPP4 (Sino Biological, 10688‐H08H) was analyzed using a BIAcore T200 instrument (Cytiva, USA).

Techniques: Derivative Assay, Binding Assay, Activation Assay, Western Blot, Phospho-proteomics, Docking Assay, Analysis, Co-Immunoprecipitation Assay, Immunofluorescence, Staining, Flow Cytometry, Cell Culture, Knockdown