Journal:

Article Title: Lipopolysaccharide-Trap-Fc, a Multifunctional Agent To Battle Gram-Negative Bacteria ▿

doi: 10.1128/IAI.00004-09

Figure Lengend Snippet: LPS-Trap-Fc1 to -Fc4 bind LPS and block LPS-mediated IL-6 production in Mono Mac 6 cells. (A) Supernatants of HEK293T cells transfected with LPS-Trap-Fc1 were incubated in the presence (lane 1) or absence (lane 3) of biotin-LPS. Additionally, supernatants were preincubated with an anti-TLR4/MD-2 MAb (lane 2) or an excess of LPS (lane 4). Complexes were immunoprecipitated with streptavidin-Sepharose, subjected to SDS-PAGE, and analyzed by Western blotting with an anti-FLAG MAb. (B) RAW 264.7 cells were incubated with LPS-Trap-Fc1 or a medium control. Cells were stimulated with 100 ng/ml LPS overnight, and IL-6 levels in supernatants were determined by ELISA. *, P < 0.002 compared to control (Mann-Whitney U test, n = 6).

Article Snippet: For specificity control, some supernatants were preincubated with either 50 μg/ml LPS ( Salmonella enterica serovar Minnesota; Sigma), which has a high binding activity for TLR4/MD-2, or 10 μg/ml anti-mouse TLR4/MD-2 MTS510 MAb (Hycult Biotechnology, Uden, The Netherlands) for 1 h at room temperature.

Techniques: Blocking Assay, Transfection, Incubation, Immunoprecipitation, SDS Page, Western Blot, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Brain research bulletin

Article Title: The effects and mechanisms of AM1241 in alleviating cerebral ischemia-reperfusion injury.

doi: 10.1016/j.brainresbull.2024.111025

Figure Lengend Snippet: Fig. 1. Effects of AM1241 on pro-inflammatory cytokines, ROS, and the MD2/TLR4 complex in BV2 cells. (A): ELISA assay for the detection of inflammatory cy tokines (TNF-α and IL-6) in BV2 cells. (B): Immunofluorescence assay to measure ROS levels in BV2 cells. (C): CO-IP to assess the impact of AM1241 on MD2/TLR4 complex. n=3, *P<0.05, **P<0.01, ***P<0.001. n=3.

Article Snippet: The respective antibodies, MD2 (11784–1-AP, Proteintech) or TLR4 (66350–1-Ig, Proteintech), were added to the supernatant and incubated overnight at 4◦C.

Techniques: Enzyme-linked Immunosorbent Assay, Immunofluorescence, Co-Immunoprecipitation Assay

Journal: Brain research bulletin

Article Title: The effects and mechanisms of AM1241 in alleviating cerebral ischemia-reperfusion injury.

doi: 10.1016/j.brainresbull.2024.111025

Figure Lengend Snippet: Fig. 2. Effects of MD2 on pro-inflammatory cytokines and ROS in BV2 cells. (A): Western blot was performed to evaluate the silencing efficiency of MD2. (B): ELISA assay for the detection of inflammatory cytokines (TNF-α and IL-6) in BV2 cells. (C): Immunofluorescence assay to measure ROS levels in BV2 cells. n=3, *P<0.05, **P<0.01, ***P<0.001. n=3.

Article Snippet: The respective antibodies, MD2 (11784–1-AP, Proteintech) or TLR4 (66350–1-Ig, Proteintech), were added to the supernatant and incubated overnight at 4◦C.

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Immunofluorescence

Journal: Brain research bulletin

Article Title: The effects and mechanisms of AM1241 in alleviating cerebral ischemia-reperfusion injury.

doi: 10.1016/j.brainresbull.2024.111025

Figure Lengend Snippet: Fig. 3. Effects of AM1241 on the MD2/TLR4 complex, inflammation, oxidative stress, and apoptosis in BV2 cells. (A): Western blot analysis to assess the efficiency of MD2 overexpression. (B): CO-IP to measure the content of MD2/TLR4 complex. (C): RT-qPCR to assess the expression levels of TNF-α, IL-6, Nox2, and BAX, C- Caspase3. (D): Western blot analysis to measure the expression levels of TNF-α, IL-6, Nox2, and BAX, C-Caspase3. n=3, *P<0.05, **P<0.01, ***P<0.001. n=3.

Article Snippet: The respective antibodies, MD2 (11784–1-AP, Proteintech) or TLR4 (66350–1-Ig, Proteintech), were added to the supernatant and incubated overnight at 4◦C.

Techniques: Western Blot, Over Expression, Co-Immunoprecipitation Assay, Quantitative RT-PCR, Expressing

Journal: Brain research bulletin

Article Title: The effects and mechanisms of AM1241 in alleviating cerebral ischemia-reperfusion injury.

doi: 10.1016/j.brainresbull.2024.111025

Figure Lengend Snippet: Fig. 5. Effects of AM1241 on MD2. (A): Molecular docking simulation predicting the binding site of AM1241 with MD2. (B): SPR assay to examine the binding of AM1241 with MD2. n=3.

Article Snippet: The respective antibodies, MD2 (11784–1-AP, Proteintech) or TLR4 (66350–1-Ig, Proteintech), were added to the supernatant and incubated overnight at 4◦C.

Techniques: Binding Assay, SPR Assay

Journal:

Article Title: Interferon regulatory factor 4 negatively regulates the production of proinflammatory cytokines by macrophages in response to LPS

doi: 10.1073/pnas.0504226102

Figure Lengend Snippet: Altered LPS signaling in IRF-4-/- macrophages. (A) Peritoneal macrophages from IRF-4+/+ and IRF-4-/- mice were stained with FITC-anti-TLR4/MD2 mAb (solid line) or without Ab (gray). (B) Peritoneal macrophages were stimulated with LPS (1 μg/ml) for 0–60 min, and the nuclear extract was prepared. Mobility-shift assay was performed with NF-κB and Oct-1 probes. (C) Peritoneal macrophages from IRF-4+/+ (open bars) and IRF-4-/- (filled bars) mice were treated with the indicated concentration of SP600125 (JNK-specific inhibitor) for 2 h and then stimulated with LPS (1 μg/ml) for an additional 24 h in the presence of the inhibitor. The relative TNF-α levels in the supernatant were expressed as the ratio to the TNF-α levels in the absence of the inhibitor. (D) Peritoneal macrophages were stimulated with LPS (1 μg/ml) for 0–60 min. Cell lysates (30 μg per lane) were separated by 12.5% SDS/PAGE and blotted. The blot was probed with anti-phospho-JNK Ab, stripped, and reprobed with anti-JNK Ab.

Article Snippet: Cells were stained with FITC-anti-F4/80 (Serotec), phycoerythrin-anti-Gr-1 (eBioscience), biotin-anti-CD11b mAb (BD Biosciences) plus avidin-Red 670 , or FITC-anti-TLR4/MD2 complex mAb (MBL, Nagoya, Japan).

Techniques: Staining, Mobility Shift, Concentration Assay, SDS Page