human recombinant toll like receptor 4 (rtlr4  (R&D Systems)

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

Journal: mBio

Article Title: Distinct immune properties of the N- and C-termini of the immunosuppressive domain of Ebola virus glycoprotein

doi: 10.1128/mbio.02278-25

Figure Lengend Snippet: TLR4 is involved in the capture of shed EBOV GP. ( A ) Binding and internalization assays on 293 cells stably expressing TLR4 (293-TLR4) and THP-1 cells were analyzed by western blotting. Cells were treated or mock-treated with recombinant TLR4 (rTLR4) and then cultured with medium or WT, mut 5, and mut 14 EBOV shed GP. Cells were then treated with trypsin to evaluate internalization of shed GP. Cell pellets were immunostained for TLR4, EBOV GP, and GAPDH as an internal control. ( B, C ) Induction of NFκB and NFAT. 293-TLR4 cells were transfected with NFAT-Luc ( B ) or NFκB-Luc ( C ), treated with CLI-095 or rTLR4 with or without CsA, treated or mock-treated with WT, mut 5, or mut 14 EBOV shed GP, and subjected to luciferase assays. Two-way ANOVA followed by a Tukey’s multiple comparison test: * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, not significant.

Article Snippet: 293T and 293-TLR4 cells were seeded at 10 5 cells per well in 12-well plates (Sigma-Aldrich), transfected with NFκB-Luc (Addgene, #111216) or NFAT-Luc (Addgene, #17870) plasmids using TransIT LT1 transfection reagent (Mirus Bio LLC) and incubated at 37°C for 48 h. Cells were then stimulated with 25 ng/mL TPA and 0.5 μM of ionomycin, or 1 μM of CsA, 10 μg/mL of rTLR4 (RnD Systems, #1478-TR-050), or 100 ng/mL CLI-095 (InvivoGen) for 1 h. Next, cells were pulsed with medium alone or with EBOV VLPs for an additional 24 h. Then, cells were lysed with Pierce Luciferase Cell lysis buffer (Thermo Fisher Scientific), and cell lysates were assayed for luciferase activity using a luminometer (Glomax 20/20, Promega).

Techniques: Binding Assay, Stable Transfection, Expressing, Western Blot, Recombinant, Cell Culture, Control, Transfection, Luciferase, Comparison

Journal: Science advances

Article Title: A neuroimmune pathway drives bacterial infection.

doi: 10.1126/sciadv.adr2226

Figure Lengend Snippet: Fig. 4. The TLR4-MYD88-MAPK3 pathway mediates CREB1 phosphorylation and function. (A) Dose-dependent impact of PKA inhibitor H-89 on ACOD1 expression post- LPS stimulation (100 ng/ml, 6 hours). (B) ACOD1 expression following LPS exposure and cotreatment with dopamine (0.5 mM) and increasing doses of PKA activator forskolin (10, 50, 100, and 200 μM). (C) Effects of cAMP analog 8-Br-cAMP on ACOD1 expression in dopamine-cotreated cells after LPS stimulation. (D) CREB1 phosphorylation and ACOD1 expression in TLR4-deficient monocytes challenged with LPS for varying durations (1 and 6 hours). (E and F) IP analyses revealing interactions within the TLR4 signal- ing complex in response to LPS and dopamine in native and DRD2-deficient monocytes. (G) Phosphorylation heatmap illustrating kinase activity shifts over time (1, 3, and 6 hours) post-LPS and dopamine treatment. (H) Effects of MAPK1/3 inhibition (VX-11e, pluripotin, ulixertinib, all 10 μM) on CREB1 and ACOD1 regulation following LPS stimu- lation. (I and J) Influence of MAPK3 knockdown or overexpression on CREB1 phosphorylation and ACOD1 expression post-LPS challenge. (K) MAPK3 activation dynamics in TLR4-knockdown THP1 cells after LPS exposure. (L) Protein expression profiling in DRD2-deficient monocytes under LPS stimulation for 1 hour. All the semiquantitative data are presented as means ± SD; n = 3 biologically independent samples. Statistical analysis was carried out using one-way ANOVA with Tukey’s multiple comparisons test.

Article Snippet: Surface plasmon resonance assay Surface plasmon resonance assay was used to investigate the interactions between recombinant DRD2 (rDRD2) and recombinant TLR4 (rTLR4) proteins (OriGene) using a Biacore system.

Techniques: Phospho-proteomics, Expressing, Activity Assay, Inhibition, Knockdown, Over Expression, Activation Assay

Journal: Science Advances

Article Title: A neuroimmune pathway drives bacterial infection

doi: 10.1126/sciadv.adr2226

Figure Lengend Snippet: ( A ) Dose-dependent impact of PKA inhibitor H-89 on ACOD1 expression post-LPS stimulation (100 ng/ml, 6 hours). ( B ) ACOD1 expression following LPS exposure and cotreatment with dopamine (0.5 mM) and increasing doses of PKA activator forskolin (10, 50, 100, and 200 μM). ( C ) Effects of cAMP analog 8-Br-cAMP on ACOD1 expression in dopamine-cotreated cells after LPS stimulation. ( D ) CREB1 phosphorylation and ACOD1 expression in TLR4 -deficient monocytes challenged with LPS for varying durations (1 and 6 hours). ( E and F ) IP analyses revealing interactions within the TLR4 signaling complex in response to LPS and dopamine in native and DRD2 -deficient monocytes. ( G ) Phosphorylation heatmap illustrating kinase activity shifts over time (1, 3, and 6 hours) post-LPS and dopamine treatment. ( H ) Effects of MAPK1/3 inhibition (VX-11e, pluripotin, ulixertinib, all 10 μM) on CREB1 and ACOD1 regulation following LPS stimulation. ( I and J ) Influence of MAPK3 knockdown or overexpression on CREB1 phosphorylation and ACOD1 expression post-LPS challenge. ( K ) MAPK3 activation dynamics in TLR4 -knockdown THP1 cells after LPS exposure. ( L ) Protein expression profiling in DRD2 -deficient monocytes under LPS stimulation for 1 hour. All the semiquantitative data are presented as means ± SD; n = 3 biologically independent samples. Statistical analysis was carried out using one-way ANOVA with Tukey’s multiple comparisons test.

Article Snippet: Surface plasmon resonance assay was used to investigate the interactions between recombinant DRD2 (rDRD2) and recombinant TLR4 (rTLR4) proteins (OriGene) using a Biacore system.

Techniques: Expressing, Phospho-proteomics, Activity Assay, Inhibition, Knockdown, Over Expression, Activation Assay