Recombinant human rage fc chimera

Three human HCC cell lines with unique metastatic characteristics were investigated. Hep3B cell line with the lowest metastasis potential [19 (link)] was purchased from Cell Bank of the Chinese Academy of Sciences (Shanghai, China); MHCC97L cell line with the moderate metastasis potential and HCCLM3 cell line with the highest metastasis potential were kindly obtained from the Liver Cancer Institute of Fudan University (Shanghai, China) [20 (link), 21 (link)]. All cell lines were maintained in DMEM medium (Gibico), supplemented with 10 % fetal bovine serum (FBS, Gibico), penicillin (100 U/ml), and streptomycin (100 μg/ml). Cells were incubated at 37 °C with 5 % CO₂, and were harvested with 0.25 % trypsin/EDTA (Gibico) in their logarithmic growth phase, washed with PBS, and resuspended in new media. The reagents used were as follows: Recombinant human HMGB1 (rhHMGB1) and recombinant human RAGE/Fc chimera, homologs to soluble form of RAGE (sRAGE), utilized as ex-RAGE were purchased from R&D systems (Inc., Minneapolis, MN, USA). The antibodies used were as follows: anti-HMGB1 neutralizing antibody, anti-RAGE neutralizing antibody (Abcam), anti-GAPDH (Cell Signal Technology), anti-NF-κB P50, anti-NF-κB P65, and goat IgG (Santa Cruz).

IMPACT Kit, restriction enzymes, Crimson Taq DNA polymerase and T4 DNA ligase were purchased from New England Biolabs (Frankfurt am Main, Germany). The 2xYT-medium was obtained from Carl Roth (Karlsruhe, Germany). HBS-P+10x Buffer was supplied by GE Healthcare (Freiburg, Germany) and recombinant human RAGE Fc chimera from a mouse myeloma cell line by R&D Systems (Wiesbaden, Germany). Calibration standards for size exclusion chromatography, MGO (40% aqueous solution), sodium pyruvate, and liquid chromatography-mass spectrometry (LC-MS)-grade acetonitrile were purchased from Sigma-Aldrich (Taufkirchen, Germany). GluC and chymotrypsin were supplied by Roche (Mannheim, Germany). 2,5-Dihydroxyacetophenone (DHAP) and protein and peptide calibration standards for matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF)-MS were obtained from Bruker Daltonik (Bremen, Germany). All other chemicals were, unless otherwise noted, purchased from Sigma-Aldrich or Acros (Geel, Belgium) and were at least of analytical grade. H₂O was purified water from a Synergi-185 laboratory water system (Millipore, Schwalbach, Germany).