Journal: Journal of orthopaedic surgery and research

Article Title: TRIM55 suppresses inflammatory response after spinal cord injury by accelerating the ubiquitination and degradation of TLR4.

doi: 10.1186/s13018-025-05922-w

Figure Lengend Snippet: Fig. 3 TRIM55 decreases the expression of TLR4 by promoting the K48-linked ubiquitination of TLR4. A The effect of TRIM55 on the protein level and ubiquitination level of TLR4 was evaluated using immunoblotting. B The interaction between TRIM55 and TLR4 proteins was analyzed by Co-IP. C PC12 cells were transfected with Flag-TRIM55, His-TLR4, and WT/K48R/K63R HA-UB. After IP with His, immunoblotting was performed with anti-HA. D PC12 cells were transfected with HA-UB, His-TLR4, and WT TRIM55 or MUT TRIM55. After IP with His, immunoblotting was performed with anti-HA. E PC12 cells were transfected with TRIM55 overexpression plasmids or empty vectors, and treated with CHX for 0, 6, 12, 18, and 24 h. Protein levels of TLR4 were detected using immunoblotting. F PC12 cells were transfected with TRIM55 overexpression plasmids or empty vectors, treated with MG132, and then stimulated with CHX for 0, 6, 12, 18, and 24 h. Protein levels of TLR4 were detected using immunoblotting. (n=3 independent biological replicates/group)

Article Snippet: To investigate TLR4 protein stability, transfected cells were first treated with MG132 (20 μM, MedChemExpress) for 8 h to suppress proteasomal activity.

Techniques: Expressing, Ubiquitin Proteomics, Western Blot, Co-Immunoprecipitation Assay, Transfection, Over Expression

Journal: Journal of orthopaedic surgery and research

Article Title: TRIM55 suppresses inflammatory response after spinal cord injury by accelerating the ubiquitination and degradation of TLR4.

doi: 10.1186/s13018-025-05922-w

Figure Lengend Snippet: Fig. 4 Overexpression of TLR4 abolishes the protective effects of TRIM55 on LPS-induced PC12 cells. A PC12 cells were transfected with TLR4 overexpression plasmids or empty vectors, and the expression of TLR4 was measured using qPCR. B-G After overexpressing TRIM55 and TLR4, PC12 cells were treated with LPS for 24 h. B The cell viability was detected with the CCK-8 assay. C-D The cell apoptosis was measured by TUNEL staining, and TUNEL-positive cells were counted. E-G The contents of IL-1β, IL-6, and TNF-α were measured by ELISA assay. All data are expressed as the means ± SD. (n=3 independent biological replicates/group)

Article Snippet: To investigate TLR4 protein stability, transfected cells were first treated with MG132 (20 μM, MedChemExpress) for 8 h to suppress proteasomal activity.

Techniques: Over Expression, Transfection, Expressing, CCK-8 Assay, TUNEL Assay, Staining, Enzyme-linked Immunosorbent Assay

Journal: Journal of Neuroscience

Article Title: Morphine Modulation of Toll-Like Receptors in Microglial Cells Potentiates Neuropathogenesis in a HIV-1 Model of Coinfection with Pneumococcal Pneumoniae

doi: 10.1523/jneurosci.0870-12.2012

Figure Lengend Snippet: Figure10. MorphineTATS.pneumoniae(luciferasetagged)inducedsynergisticincreaseinbacterialdisseminationand proinflammatorycytokinesaresignificantlyattenuatedinTLR2,4knock-outsandTLR2/4doubleknock-outmice.A,Asignificant decrease in bacterial dissemination into the CNS of TLR 2, 4 knock-outs and TLR2/4 double knock-out mice following morphine, TAT, and S. pneumoniae lysate treatment. B, Quantification of bacterial dissemination in the brain tissue of WT, TLR2KO, and TLR4KO mice. Data represents mean SEM of three independent experiments. *p 0.01, ***p 0.001. C, To determine apoptosis,animals(6pergroup)weretreatedasdescribedinFigure4Aandkilledat72h.Brainswereremovedandsnapfrozenin liquid nitrogen. Cryostat sections (5 m) were used to evaluate apoptosis using TUNEL staining (Intergen) according to the manufacturer’s instruction. DAPI staining shows the nuclei of cells. Apoptosis is significantly lower in the TLR knock-out animals compared with wild-type following treatment with morphine, TAT, and S. pneumoniae (S.p.) lysate. Scale bars, 10 m. D, Synergistic increase in proinflammatory cytokines in WT mice following morphine, TAT, and S. pneumoniae lysate treatment is significantly attenuated in brain homogenate harvested from TLR 2, 4 knock-outs and TLR2/4 double knock-out mice. Data representsmeanSEMofthreeindependentexperiments.*p0.01.E,SurvivalcurveofWTandTLR2,4knock-outsandTLR2/4 double knock-out mice treated with morphine TAT S. pneumoniae. Animals were treated as described in Figure 4A and survival followed for 5 d. Data represents mean SEM of three independent experiments.

Article Snippet: Primer sequences used in the amplification of TLR genes are TLR4 (accession no. NM_021297), TLR2 (accession no. NM_011905), and TLR9 (accession no. NM_031178) (all from Origene Techonologies).

Techniques: Knock-Out, TUNEL Assay, Staining

Journal: EBioMedicine

Article Title: ACE2-independent SARS-CoV-2 virus entry through cell surface GRP78 on monocytes - evidence from a translational clinical and experimental approach.

doi: 10.1016/j.ebiom.2023.104869

Figure Lengend Snippet: Fig. 1: Expression of cell surface markers on CD14+ cells and soluble factors in blood plasma in controls (n = 8) and COVID-19 patients (n = 8). Expression of csGRP78 (a) and CD86 (b) on monocytes. Values represent percentages of surface marker positive CD14+ cells. Presented humoral factors are soluble GRP78 (sGRP78) (c) and cytokines IFN-γ, IL-6, IL-10 and sIL-2Ra (d). Values represent concentration in pg/ml (control n = 5). Violine plots indicate median values and interquartile range (IQR). Statistical differences between groups were calculated by Mann–Whitney U test *P < 0.05, **P < 0.01, ***P < 0.005.

Article Snippet: Except for GRP78 (14464, Cell Signaling Technology, Danvers, MA, USA), TLR2 (130-120-052, Miltenyi Biotec, Bergisch Gladbach, Germany), and TLR4 (FAB6248C, R&D Systems, Minneapolis, MN, USA), all antibodies were obtained from BD Biosciences (CD14: 345784, CD40: 555589, CD69: 340548, CD80: 557227, CD86: 555665, HLA-DR: 555812, Franklin Lakes, NJ, USA).

Techniques: Expressing, Clinical Proteomics, Marker, Concentration Assay, Control, MANN-WHITNEY

Journal: EBioMedicine

Article Title: ACE2-independent SARS-CoV-2 virus entry through cell surface GRP78 on monocytes - evidence from a translational clinical and experimental approach.

doi: 10.1016/j.ebiom.2023.104869

Figure Lengend Snippet: Fig. 4: GRP78 is expressed in CD14+ monocytes and CD68+ macrophages in lung tissue of COVID-19 patients. Representative staining of CD14 (yellow) and GRP78 (red) (a) and CD68 (yellow) and GRP78 (red) (b). DNA counterstain was performed with DAPI (blue). Magnification was 20× (scale bar 100 μm) with inlet (magnification 60× (scale bar 30 μm)). Violin plot showing number of CD14+/GRP78+ cells per high power field (HPF) of gender and age matched patients who died from myocardial infarction (control) and of COVID-19 (c). Representative staining from CD14 (red) and SARS-CoV-2 (yellow) in samples from COVID-19 patients (d), DNA counterstain was performed with DAPI (blue). Magnification was 20× (scale bar 100 μm) with inlet (magnification 60× (scale bar 30 μm)). Values are presented as median (IQR). Statistical differences between groups were calculated by Mann–Whitney U test ****P < 0.001.

Article Snippet: Except for GRP78 (14464, Cell Signaling Technology, Danvers, MA, USA), TLR2 (130-120-052, Miltenyi Biotec, Bergisch Gladbach, Germany), and TLR4 (FAB6248C, R&D Systems, Minneapolis, MN, USA), all antibodies were obtained from BD Biosciences (CD14: 345784, CD40: 555589, CD69: 340548, CD80: 557227, CD86: 555665, HLA-DR: 555812, Franklin Lakes, NJ, USA).

Techniques: Staining, Control, MANN-WHITNEY

Journal: EBioMedicine

Article Title: ACE2-independent SARS-CoV-2 virus entry through cell surface GRP78 on monocytes - evidence from a translational clinical and experimental approach.

doi: 10.1016/j.ebiom.2023.104869

Figure Lengend Snippet: Fig. 5: Interaction and colocalization between csGRP78 and SARS-CoV-2 spike protein. Surface plasmon resonance analysis showing the interaction intensity between GST-GRP78 and spike protein expressed as affinity constant KD = 55.2 nM (a). GST Pull-down assay showing direct interaction between GST-GRP78 with SARS-CoV-2 spike protein (b) and S1 subunit (c). Co-immunoprecipitation demonstrating GRP78 extraction together with SARS-CoV-2 S1 subunit (lane 2) and spike protein (lane 4) on THP-1 cell membrane (d). Cytokine cocktail (IL-1β, IL-6, TNF and IFN-γ) induced csGRP78 expression (MFI) on CD14+ monocytes derived from PBMCs from healthy donors (n = 3) (e) and on THP-1 cells (n = 3) (f) over time course of 48 h. Representative immunofluorescence staining from triplicates of SARS-CoV-2 spike protein (green) and csGRP78 (red) in THP-1 cells before and after cytokine cocktail stimulation for 48 h. Arrows indicate colocalization. Scale bar 10 μm (g). Flow- cytometric analysis of spike protein adhesion (upper chart), and csGRP78 expression (lower chart) on THP-1 cells after 48 h spike protein stimulation with or without cytokine cocktail, brefeldin A (0.03 μg/ml) or AR-12 (5 μM), presented as fold change (h). Violine plots indicate median values and interquartile range. Statistical differences between two groups were calculated by Mann–Whitney U test and between four groups by ANOVA with Tukey multiple comparison test, *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001.

Article Snippet: Except for GRP78 (14464, Cell Signaling Technology, Danvers, MA, USA), TLR2 (130-120-052, Miltenyi Biotec, Bergisch Gladbach, Germany), and TLR4 (FAB6248C, R&D Systems, Minneapolis, MN, USA), all antibodies were obtained from BD Biosciences (CD14: 345784, CD40: 555589, CD69: 340548, CD80: 557227, CD86: 555665, HLA-DR: 555812, Franklin Lakes, NJ, USA).

Techniques: SPR Assay, Pull Down Assay, Immunoprecipitation, Extraction, Membrane, Expressing, Derivative Assay, Staining, MANN-WHITNEY, Comparison

Journal: bioRxiv

Article Title: Extract from Polygala fallax Hemsl. Protects Kidneys in db/db Mice by Inhibiting the TLR4/MyD88/NF- κ B Signaling Pathway

doi: 10.1101/2023.08.08.552432

Figure Lengend Snippet: The effect of EPF on the levels of TLR4, p-NF- κ B p65, MyD88 and MMP-9 in the kidney of db/db mice. (A) Immunohistochemical map of TLR4, NF- κ B p65, MyD88 and MMP-9 (400×) (B) Imm unohistochemical optical density analysis of TLR4, NF- κ B p65, MyD88and MMP-9 proteins in kidne y tissue (n=3). All data are expressed as mean ± S.E.M. ##P<0.01 compared with the control group; *P<0.05, **P<0.01 compared with the DN group.

Article Snippet: Then, the corresponding primary antibodies were added, including TLR4 (CST, Boston, USA) (1:1400), MyD88 (CST, Boston, USA) (1:1400), NF- κ B p65 (CST, Boston, USA) (1:1400), and MMP-9 (CST, Boston, USA) (1:1000), and the membrane was incubated overnight at 4°C. β -actin (1:5000) was used as an internal referen ce.

Techniques: Immunohistochemical staining, Control

Journal: bioRxiv

Article Title: Extract from Polygala fallax Hemsl. Protects Kidneys in db/db Mice by Inhibiting the TLR4/MyD88/NF- κ B Signaling Pathway

doi: 10.1101/2023.08.08.552432

Figure Lengend Snippet: The effect of EPF on the TLR4/MyD88/NF- κ B signaling pathway in the kidney tissue of db/db mice. (A) Representative bands of TLR4,MMP-9, NF- κ B p65, p-NF- κ B p65 and MyD88 obtained by western blotting. (B) The relative expression of TLR4, MMP-9, p-NF- κ B p65/NF- κ B p65 and My D88 proteins (n=3). (C) The relative expression of TLR4, MMP-9, NF- κ B p65, and MyD88 mRNA (n=3). All data are expressed as mean ± S.E.M. ## P<0.01 compared with the control group; *P<0.05, **P<0.01 compared with the DN group.

Article Snippet: Then, the corresponding primary antibodies were added, including TLR4 (CST, Boston, USA) (1:1400), MyD88 (CST, Boston, USA) (1:1400), NF- κ B p65 (CST, Boston, USA) (1:1400), and MMP-9 (CST, Boston, USA) (1:1000), and the membrane was incubated overnight at 4°C. β -actin (1:5000) was used as an internal referen ce.

Techniques: Western Blot, Expressing, Control