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: International journal of molecular sciences

Article Title: Integrated Metabolomics and Network Pharmacology to Reveal the Mechanisms of Forsythia suspensa Extract Against Respiratory Syncytial Virus.

doi: 10.3390/ijms26115244

Figure Lengend Snippet: Figure 7. IHC images showing the protein levels of TLR4 (A) and p38 MAPK (B) in mouse lung tissues induced by RSV and inhibited by Forsythia suspensa (400× magnification).

Article Snippet: Reagents Methanol (Fisher Chemical, Fair Lawn, NJ, USA); acetonitrile (Fisher Chemical, USA); formic acid (CNW, Düsseldorf, Germany); isopropanol (Merck, Darmstadt, Germany); L-2-chlorophenylalanine (≥98%, Adamas Reagent, Riehen, Switzerland); Milli-Q ultrapure water; DMSO (AMRESCO, Solon, OH, USA); PBS-P (Cytiva, USA); HBS-EP (Cytiva, USA); Amine Coupling Kit (Cytiva, USA); isoflurane anesthesia (RWD, China); TLR4 protein (R&D Systems, Minneapolis, MN, USA); p38 α-MAPK14 protein (BPS Bioscience Inc., Santiago, Chile); resatorvid (MCE, Monmouth Junction, NJ, USA); SB 202190 (MCE, USA); DAB chromogenic reagent (Servicebio, Wuhan, China); primary antibodies (anti-p38 rabbit pAb and Anti-TLR4 Rabbit pAb, Servicebio, China); and secondary antibody G1213-100UL (Servicebio, China).

Techniques:

Journal: International journal of molecular sciences

Article Title: Integrated Metabolomics and Network Pharmacology to Reveal the Mechanisms of Forsythia suspensa Extract Against Respiratory Syncytial Virus.

doi: 10.3390/ijms26115244

Figure Lengend Snippet: Figure 8. Interaction of FS pharmacodynamic components with core targets. (A) AKT1 with Astraglin; (B) CASP8 with Rutin; (C) IL6 with Astraglin; (D) MAPK8 with Wogonin; (E) RELA with Astraglin; (F) TNF with Quercetin; (G) p38α·MAPK14 with Rutin; (H) TLR4 with Rutin.

Article Snippet: Reagents Methanol (Fisher Chemical, Fair Lawn, NJ, USA); acetonitrile (Fisher Chemical, USA); formic acid (CNW, Düsseldorf, Germany); isopropanol (Merck, Darmstadt, Germany); L-2-chlorophenylalanine (≥98%, Adamas Reagent, Riehen, Switzerland); Milli-Q ultrapure water; DMSO (AMRESCO, Solon, OH, USA); PBS-P (Cytiva, USA); HBS-EP (Cytiva, USA); Amine Coupling Kit (Cytiva, USA); isoflurane anesthesia (RWD, China); TLR4 protein (R&D Systems, Minneapolis, MN, USA); p38 α-MAPK14 protein (BPS Bioscience Inc., Santiago, Chile); resatorvid (MCE, Monmouth Junction, NJ, USA); SB 202190 (MCE, USA); DAB chromogenic reagent (Servicebio, Wuhan, China); primary antibodies (anti-p38 rabbit pAb and Anti-TLR4 Rabbit pAb, Servicebio, China); and secondary antibody G1213-100UL (Servicebio, China).

Techniques:

Journal: International journal of molecular sciences

Article Title: Integrated Metabolomics and Network Pharmacology to Reveal the Mechanisms of Forsythia suspensa Extract Against Respiratory Syncytial Virus.

doi: 10.3390/ijms26115244

Figure Lengend Snippet: Figure 9. Biacore interaction assays measuring the binding of target proteins TLR4 (A) and p38α·MAPK14 (B) to components of FS. Note: The interaction results were analyzed using the kinetics and affinity method in the Kinetics Wizard template. The curves in the figure, from top to bottom, represent the response values of drugs passing over the target protein surface at decreasing concentrations.

Article Snippet: Reagents Methanol (Fisher Chemical, Fair Lawn, NJ, USA); acetonitrile (Fisher Chemical, USA); formic acid (CNW, Düsseldorf, Germany); isopropanol (Merck, Darmstadt, Germany); L-2-chlorophenylalanine (≥98%, Adamas Reagent, Riehen, Switzerland); Milli-Q ultrapure water; DMSO (AMRESCO, Solon, OH, USA); PBS-P (Cytiva, USA); HBS-EP (Cytiva, USA); Amine Coupling Kit (Cytiva, USA); isoflurane anesthesia (RWD, China); TLR4 protein (R&D Systems, Minneapolis, MN, USA); p38 α-MAPK14 protein (BPS Bioscience Inc., Santiago, Chile); resatorvid (MCE, Monmouth Junction, NJ, USA); SB 202190 (MCE, USA); DAB chromogenic reagent (Servicebio, Wuhan, China); primary antibodies (anti-p38 rabbit pAb and Anti-TLR4 Rabbit pAb, Servicebio, China); and secondary antibody G1213-100UL (Servicebio, China).

Techniques: Binding Assay