recombinant human md2 rhmd2 protein  (R&D Systems)

Journal: Exploration

Article Title: Ganoderic Acids Alleviate Neuroinflammation by Targeting Myeloid Differentiation Factor 2 for Ischemic Stroke Therapy

doi: 10.1002/EXP.20240147

Figure Lengend Snippet: Interaction of GA monomers with the MD2/TLR4 complex. (A–H) Surface plasmon resonance (SPR) analysis showing direct binding of Ganoderic acid A, B, C2, C6, G, H, K, and Ganoderenic acid B to MD2. (I) Binding of GA‐A to MD2 as assessed by protein microarray analysis. (J) Identification of MD2/TLR4 complexes via immunoprecipitation.

Article Snippet: To investigate the molecular interaction between GA monomers and MD2, recombinant human MD2 (rhMD2) protein (R&D Systems) was employed.

Techniques: SPR Assay, Binding Assay, Microarray, Immunoprecipitation

Journal: Exploration

Article Title: Ganoderic Acids Alleviate Neuroinflammation by Targeting Myeloid Differentiation Factor 2 for Ischemic Stroke Therapy

doi: 10.1002/EXP.20240147

Figure Lengend Snippet: Molecular docking of GA‐K with MD2 and its effect on cerebral ischemic injury in the mouse tMCAO model. (A) Molecular docking of GA‐K (yellow) with the MD2 protein (green), analyzed using the Trips molecular modeling software. (B) Representative coronal brain sections stained with TTC, showing typical infarct areas in white. Scale bar = 5 mm. (C) Quantification of infarct volume. (D) Neurological deficit scores quantification. The data are presented as the mean ± SEM ( n = 8). Statistical significance: ** p < 0.01 compared to the tMCAO group.

Article Snippet: To investigate the molecular interaction between GA monomers and MD2, recombinant human MD2 (rhMD2) protein (R&D Systems) was employed.

Techniques: Software, Staining

Journal: Exploration

Article Title: Ganoderic Acids Alleviate Neuroinflammation by Targeting Myeloid Differentiation Factor 2 for Ischemic Stroke Therapy

doi: 10.1002/EXP.20240147

Figure Lengend Snippet: GAs suppress MD2/TLR4 complex formation and inhibit MAPK and NF‐κB signaling pathways in a mouse model of tMCAO. GA (administered at doses of 0 or 20 mg kg −1 , i.p.) was given immediately after reperfusion. At 24 h post‐reperfusion, total and nuclear proteins were isolated from the cortical penumbra for analysis by Western blotting. (A) Immunoprecipitation analysis of the MD2/TLR4 complex in the ischemic hemisphere. (B) Quantification of MD2 expression levels. (C) Representative Western blot images showing proteins involved in the MAPK signaling pathway. (D) Quantitative analysis of phosphorylation levels. (E) Representative Western blot images of nuclear NF‐κB and AP‐1. (F) Quantification of protein expression. The data are presented as the mean ± SEM ( n = 4). Statistical significance is indicated as follows: ### p < 0.001 compared to the sham group, * p < 0.05, ** p < 0.01 compared to the vehicle‐treated tMCAO group.

Article Snippet: To investigate the molecular interaction between GA monomers and MD2, recombinant human MD2 (rhMD2) protein (R&D Systems) was employed.

Techniques: Protein-Protein interactions, Isolation, Western Blot, Immunoprecipitation, Expressing, Phospho-proteomics

Journal: Exploration

Article Title: Ganoderic Acids Alleviate Neuroinflammation by Targeting Myeloid Differentiation Factor 2 for Ischemic Stroke Therapy

doi: 10.1002/EXP.20240147

Figure Lengend Snippet: MD2 knockout reduces microglia activation and improves acute cerebral ischemic injury in the tMCAO mouse model. (A) Representative micrographs (magnification ×100) showing immunofluorescent staining of MD2 (red) in the peri‐infarct area of the cortex and the dentate gyrus of the hippocampus, 24 h after reperfusion. Scale bars: 50 µm. WT and MD2‐KO mice underwent 1 h of tMCAO, followed by 24 h of reperfusion. GA (0 or 20 mg kg −1 , i.p.) was administered immediately post‐reperfusion. (B) Representative micrographs depicting immunofluorescence for Iba‐1 (green). Primary microglial cells were isolated from WT and MD2‐KO mice, pretreated with GA (50 µg mL −1 ) or vehicle for 1 h, then stimulated with LPS (10 ng mL −1 ) for 12 h. (C) Representative Western blots illustrating levels of p‐JNK, p‐ERK, p‐P38, and p‐NF‐κB. (D) Representative Western blots for inflammatory mediators iNOS, COX‐2, and TNF‐α ( n = 4). (E) Representative coronal brain sections stained with TTC. Infarct areas appear white. Bar = 5 mm. (F) Infarction volume assessment. (G) Neurological deficit score quantification. The data are presented as the mean ± SEM ( n = 8). Statistical significance is indicated as follows: * * P < 0.01, ** * P < 0.001 compared to the WT tMCAO group.

Article Snippet: To investigate the molecular interaction between GA monomers and MD2, recombinant human MD2 (rhMD2) protein (R&D Systems) was employed.

Techniques: Knock-Out, Activation Assay, Staining, Immunofluorescence, Isolation, Western Blot

Journal: Exploration

Article Title: Ganoderic Acids Alleviate Neuroinflammation by Targeting Myeloid Differentiation Factor 2 for Ischemic Stroke Therapy

doi: 10.1002/EXP.20240147

Figure Lengend Snippet: Proposed mechanism of GA in alleviating cerebral ischemic injury. GA monomers interact directly with MD2, preventing the dimerization of MD2 and TLR4, as well as the subsequent activation of downstream MAPK and NF‐κB signaling pathways. This process lowers inflammatory mediator production and reduces microglial overactivation.

Article Snippet: To investigate the molecular interaction between GA monomers and MD2, recombinant human MD2 (rhMD2) protein (R&D Systems) was employed.

Techniques: Activation Assay, Protein-Protein interactions

Journal: ACS Nano

Article Title: Force Nanoscopy Demonstrates Stress-Activated Adhesion between Staphylococcus aureus Iron-Regulated Surface Determinant Protein B and Host Toll-like Receptor 4

doi: 10.1021/acsnano.4c12648

Figure Lengend Snippet: IsdB-TLR4 strength confirmed by anti-TLR4 blocking and S. aureus strain lacking IsdB. (a) Rupture force histograms of 2 representative Δ spa cells, cultured in RPMI medium, obtained by recording force–distance curves in PBS at a retraction speed of 1 μm/s between TLR4-functionalized AFM tips before ( n = 188 and 276 adhesive curves, for cell #1 and cell #2, respectively) and after blocking with 100 μg/mL of anti-TLR4 monoclonal antibody ( n = 98 and 65 adhesive curves, for cell #1 and cell #2, respectively). (b) Rupture force and rupture length histograms of a representative S. aureus cell expressing IsdB (WT, n = 200 adhesive curves) cultured in RPMI. (c) Force data of a representative S. aureus cell lacking IsdB (Δ isdB , n = 95 adhesive curves) cultured in RPMI. Schemes of the SMFS setups and representative retraction force profiles are shown as insets. Box plots comparing (d) binding frequency and (e) rupture forces obtained for Δ spa cells before and after blocking with anti-TLR4 monoclonal antibody, after tip treatment with monoclonal mouse IgG as a negative control, WT and Δ isdB strains ( n = 8, 8, 10, 13, and 9 cells, respectively). Means are represented by stars, medians by lines, boxes indicate the 25–75% quartiles, and whiskers the standard deviation. P -values were determined using Kruskal–Wallis test followed by post hoc Dunn’s test.

Article Snippet: For blocking experiments, TLR4-functionalized cantilevers were exposed to a 100 μg/mL anti-TLR4 monoclonal antibody (MAB14782, R&D Systems) solution in PBS for 1 h. A monoclonal mouse IgG antibody (Human MD-2 Antibody, MAB1787, R&D Systems) was used as negative control.

Techniques: Blocking Assay, Cell Culture, Adhesive, Expressing, Binding Assay, Negative Control, Standard Deviation