TLR9 Antibody - #DF2970

Fig. 5: NETs promoted STING upregulation by activating TLR2 signaling. A KEGG analysis identified significantly altered pathways in control and NET-treated HUVECs. B Relative mRNA levels of TLRs in control and NET-treated HUVECs (n = 3). C Western blot images of TLRs expression in control and NET-treated HUVECs. D Representative images of immunofluorescence staining of TLR2 in control and NET-treated HUVECs. Scale bar: 30 μm. The expression of TLR2 was assessed by fluorescence intensity (n = 3). E The cell viability of HUVECs pretreated with C-29 (n = 3). F Relative mRNA levels of TLR2, STING, and TF in HUVECs pretreated with C-29 (n = 3). G Western blot images of STING pathway and TF expression in HUVECs pretreated with C-29. H Relative mRNA levels of STING in HUVECs transfected with LV-NC-RNAi or LV-TLR2-RNAi (n = 3). I Western blot images of TLR2 expression in HUVECs transfected with LV-NC-RNAi or LV-TLR2-RNAi. J Western blot was performed to analyze the levels of STING and TF expression in HUVECs transfected with LV-NC-RNAi or LV-TLR2-RNAi under stimulation of NETs. K Western blot images of TLR2 expression in HUVECs pretreated with DNase I. L Western blot images of TLR2 expression in murine lung tissues treated with DNase I. Each bar represents the mean ± SD. The comparison between the two groups was performed using unpaired t-tests (B, D, and H). Statistical analysis for three or more groups was carried out using 1-way ANOVA (E and F). *p 

FIGURE 4 Cytoplasmic mtDNA markedly activated STING signalling. BMDCs were treated with mtDNA (10 μg/ml), or transfected with vehicle or mtDNA (10 μg/ml) for 24 h, then were harvested for western blot and qPCR. (A) Representative blots and relative quantification protein levels of STING pathway (n = 3). (B) qPCR quantitation of IFN‐β (n = 7). (C) Representative images of nuclear translocation of IRF3 in BMDC treated as indicated. Scale bar: 5 μm. (D) Representative blots and relative quantification protein levels of TLR9 pathway (n = 3). (E) qPCR quantitation of TNF‐α, IL‐6 and VCAM‐1 (n = 6–7). (F) Representative blots and relative quantification protein levels of NLRP3 pathway (n = 3). (G) qPCR quantitation of IL‐1β, IL‐18 (n = 7). Data are shown as the mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001. BMDC, bone marrow derived dendritic cell; IFN, interferon; mtDNA, mitochondrial DNA; mtDNA (T), mtDNA transfection; Veh (T), vehicle transfection.

FIGURE11 MENKupregulatedTLRinRAW264.7cellsandmodulatedcytokineproduction. (A)WesternblotanalysisofTLR7,TLR8,TLR9, IL-1b,TNF-a,andIL 10proteinexpressionlevelsintheControl,SFTSV,andMENK–SFTSVgroups. (B)Histogramsofthegrayscaleanalysisofthecorrespondingproteins. Controlgroupvs.SFTSVgroupandSFTSVgroupvs.MENK-SFTSVgroup,*P

Fig 6. |TLR3 mediates the MHV-68-induced SOCS1 production. (A) BMMs were transfected with siRNAs against Tlr2, Tlr3, Tlr4, Tlr7, Tlr9 (si-TLR) or an irrelevant target (si-Control), respectively. Forty-eight hours post transfection, cells were infected with MHV-68 at MOI = 10. At 8 hpi, RNA and protein were then extracted. RT-qPCR was performed to determine SOCS1 mRNA levels and western blotting was performed to determine the protein expression levels of SOCS1 and the individual TLRs, respectively. The SOCS1 mRNA levels are expressed as values relative to the MHV-68 infected, si-Control transfected cells.

Figure 3. Cytosolic mtDNA stress enhances the secretion of IL-6 by activating the nuclear factor-κB (NF-κB) signaling pathway A-B. qRT-PCR analysis for the mRNA expression of genes encoding TAM recruitment associated cytokines and chemokines including CCL2, CSF1, TGF-β, IL-6, and CCL22 in OVISE (a) and CAOV4 (b) cells 48 h after lentiviral mediated TFB2M overexpression. C-D. qRT-PCR (c) analysis of IL-6 transcription in cells and ELISA (d) assay of IL-6 secretion in the supernatants of cultured OVISE and CAOV4 cells with TFB2M overexpression alone, treated with DNase I alone or with combined TFB2M overexpression and DNase I treatment. E. qPCR was used to determine the copy number of mtDNA in the cytoplasm of OVISE and CAOV4 cells with TFB2M overexpression alone, treated with DNase I alone or with combined TFB2M overexpression and DNase I treatment. F. Immunofluorescent staining of TLR9 in OVISE and CAOV4 cells with or without TFB2M overexpression. The interactions between TLR9 and cytosolic mtDNA were indicated by white arrows. G-J. Western blot analyses were performed to detect the protein levels of TFB2M in whole cells and phospho-NF-κB p65 (p-p65) (Ser536) and p65 in the cytoplasm and nucleus of OVISE and CAOV4 cells with TFB2M overexpression alone or with combined ODN INH-18 (a TLR9 antagonist), DNase I or PDTC (an NF-κB inhibitor) treatment. Total TFB2M, nucleus p-p65, and p65 in each group were quantified using ImageJ software (h-j). Data shown are the mean ± SD. from three independent experiments. EV: empty vector. **p < 0.01, ***p < 0.001, n.s., not significant.