Fluorescein isothiocyanate labelled lps fitc lps

25HC, LPS (E. coli O111:B4) and fluorescein isothiocyanate‐labelled LPS (FITC‐LPS) were purchased from Sigma‐Aldrich (St. Louis, MO, USA). Recombinant human MD‐2 (rhMD‐2) protein was obtained from R&D Systems (Minneapolis, MN, USA). Biotin‐LPS was purchased from Invivogen (San Diego, CA, USA). Anti‐MD‐2 antibody was from Abcam (Cambridge, UK). Antibodies against phospho‐Akt, phospho‐NF‐κB p65, phospho‐JNK mitogen‐activated protein kinase (MAPK), phospho‐Erk42/44 MAPK, phospho‐p38 MAPK, Akt, NF‐κB p65, MAPKs of JNK, Erk42/44, p38, and β‐actin were purchased from Cell Signaling Technology (Boston, MA, USA).

Compound L2H21 was provided by our laboratory and purified using HPLC with a purity of 99.3%. In in vitro experiments, L2H21 was dissolved in dimethyl sulphoxide (DMSO) solution and similar volume of DMSO followed as a vehicle control. In the in vivo mortality study, L2H21 was initially dissolved in water with macrogol 15 hydroxystearate (a non‐ionic solubilizer for injection from BASF) in water. The concentration of L2H21 and solubilizer was 2 mg/ml and 8% in water solution, respectively. For the vehicle, the solubilizer was prepared at 8% in water. In the in vivo ALI study, L2H21 was resuspended in 0.5% CMC‐Na solution. LPS, fluorescein isothiocyanate‐labelled LPS (FITC‐LPS) and Pam3CK were purchased from Sigma‐Aldrich (St. Louis, MO, USA). Anti‐CD68, anti‐MD‐2 and anti‐TLR4 antibodies were purchased from Santa Cruz (Santa Cruz, CA, USA). Recombinant human MD‐2 (rhMD‐2) protein was purchased from R&D Systems, Inc. (Minneapolis, MN, USA). Mutated rhMD‐2 protein was obtained by the methods described in our previous publication 19.