recombinant human md-2 (rhmd-2, or rhmd-2 mutant) proteins  (Biowit Technologies)

Journal: Journal of Cellular and Molecular Medicine

Article Title: 25‐Hydroxycholesterol protects against acute lung injury via targeting MD ‐2

doi: 10.1111/jcmm.13820

Figure Lengend Snippet: 25 HC prevents LPS binding to TLR 4 receptor through interacting with MD ‐2. (A) After anti‐ MD ‐2 antibody was coated in 96‐well plates for overnight at 4 °C, the rh MD ‐2 (0.1 μmol/L) and biotin‐ LPS (100 ng/ mL ) were added into the wells in the presence or absence of 25 HC (1, 5, 10, or 20 μmol/L) for 30 min. The binding of LPS to MD ‐2 was indicated as absorbance values at 450 nm by ELISA . **P < 0.01 vs biotin‐ LPS group. (B) The fluorescence emission spectra of rh MD ‐2 (5 nmol/L) were detected with or without 25 HC (1, 5, 10, or 20 μmol/L). (C) RAW 264.7 cells were pretreated at various concentrations of 25 HC (1, 5, 10, or 20 μmol/L) for 2 h and then treated with FITC ‐ LPS (2 μg/mL) for 20 min. The cells were analysed by flow cytometry. *P < 0.05 and **P < 0.01 vs FITC ‐ LPS group. (D) RAW 264.7 cells were pretreated with either 25 HC (10 μmol/L) or 0.1% ethanol (EtOH) for 2 h and then treated with Alexa Fluor 568‐conjugated LPS (6 μg/ mL ) for 15 min. Confocal immunofluorescence microscopy was performed for analysis. Scale bar, 10 μm. (E) Molecular docking of 25 HC with MD ‐2 protein was conducted by AutoDock program. 25 HC in purple red and lipid IV a in yellow were represented with sticks. MD ‐2 was shown as the solid surface (left). The refined model was rendered as 25 HC by purple red and its interacting residues on MD ‐2 by dark blue. A hydrogen bond between 25 HC and Tyr‐102 residue of MD ‐2 was depicted as a dotted line (right). Quantitative data are shown as mean ± SEM

Article Snippet: Recombinant human MD‐2 (rhMD‐2) protein was obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Fluorescence, Flow Cytometry, Immunofluorescence, Microscopy, Residue

Journal: Journal of Cellular and Molecular Medicine

Article Title: 25‐Hydroxycholesterol protects against acute lung injury via targeting MD ‐2

doi: 10.1111/jcmm.13820

Figure Lengend Snippet: Proposed model of 25 HC preventing LPS ‐induced TLR 4 signalling pathway. LPS binding to MD 2 promotes the dimerization of TLR 4/ MD ‐2. The conformational changes in the TLR 4 induce the recruitment of intracellular adaptor proteins to activate the downstream signalling pathway. The MyD88‐dependent pathway involves activation of MAPK cascades and IKK (IκB kinase). Phosphorylation of IKK s leads to degradation of IκBα and the release of NF ‐κB. Transcription factors such as NF ‐κB, AP ‐1 and etc. participate in driving gene expression of proinflammatory cytokines. The activated PI 3K/Akt in the downstream of TLR 4 induces nuclear translation of NF ‐κB. 25 HC could directly interact with MD 2 to prevent LPS ‐induced activation of Akt and NF ‐κB signal pathway

Article Snippet: Recombinant human MD‐2 (rhMD‐2) protein was obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Binding Assay, Activation Assay, Expressing