Tgf β neutralizing antibody

Cells were cultured at 37 °C under 5% CO₂. The human THP-1 cell line was purchased from Riken Bioresource Center (RCB1189). THP-1 cells were cultured in RPMI medium with 10% fetal bovine serum (FBS). THP-1 macrophages were generated by incubating the cells with 100 nmol/L of phorbol-myristate-acetate (PMA) for 24 h. Raw 264.7 cells were purchased from ATCC and cultured in DMEM with 2 mM Glutamine and 10% Foetal Bovine Serum (FBS). Recombinant PTX3 (rPTX3) was provided by Perseus Proteomics Inc. (Tokyo, Japan). It was made by the procedure that was written in the article [12] . Human umbilical vein endothelial cells (HUVECs) and specific medium for HUVEC culture were obtained from Lonza (Basel, Swiss). Experiments using HUVECs were conducted with cells between passage numbers three and five (P3–P5). Human monocyte-derived macrophages were generated from healthy volunteers' blood.
Peripheral blood mononuclear cells (PBMC) were separated using Ficoll-Paque PLUS solution and lymphocytes were washed away after monocyte adhesion on tissue culture-treated culture plates. Cells were cultured with 20 ng/mL of granulocyte macrophage-colony stimulating factor (GM-CSF, Pepro Tech, Rocky Hill, NJ) in RPMI medium containing 10% FBS and were incubated for 6 days prior to assaying. TGF-β neutralizing antibody was obtained from R&D Systems (Minneapolis, MN. Ab-100-NA rabbit polyclonal, 10 µg/mL).

Recombinant human TGF-β1 was purchased from PeproTech (Princeton, NJ, USA). SB431542 was purchased from Selleckchem (Houston, TX, USA). RGD peptide was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). TGF-β neutralizing antibody was purchased from R&D Systems (Minneapolis, MN, USA). Anti-TNC antibodies for IHC staining and western blot analysis were purchased from Santa Cruz Biotechnology and Abcam (Cambridge, UK), respectively. Anti-GAPDH antibody was from AbClone (Seoul, Korea). Anti-SMAD2 (SMAD family member 2), anti-phospho-SMAD2, anti-SMAD3, and anti-phospho-SMAD3 antibodies were from Cell Signaling Technology (Danvers, MA, USA). Anti-pro-COL1A1 and anti-MMP-1 antibodies have been described previously [49 (link)]. Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG, goat anti-rabbit IgG, and rabbit anti-goat IgG antibodies were purchased from KOMA Biotech (Seoul, Korea). Rhodamine Red-X-conjugated goat anti-mouse IgG and Alexa Fluor 488-conjugated rabbit anti-goat IgG antibodies were purchased from Invitrogen (Carlsbad, CA, USA).

Recombinant human TSP-4 protein (rh-TSP-4), recombinant human TGF-β1 protein and a TGF-β-neutralizing antibody were obtained from R&D Systems (Minneapolis, MN, USA). An integrin α2 neutralizing antibody was purchased from Millipore (Darmstadt, Germany). LY294002 was obtained from Selleckchem (Houston, TX, USA). Detailed information for the antibodies used in the present study is displayed in Additional file 1: Table S1. All reagents were stored following the manufacturer’s instructions.