Tgf β1 neutralizing antibody

All cell lines were purchased from Cell Bank of Type Culture Collection of Chinese Academy of Sciences, Shanghai Institute of Cell Biology, Chinese Academy of Sciences. Cells were cultured in RPMI 1640 or D-MEM supplemented with 10% fetal bovine serum (FBS) (Cat# 16000-044, Gibco) at 37°C in a humidified atmosphere containing 5% CO₂.
GFAT1 antibody (Cat# ab176775) was purchased from Abcam (Cambridge, MA, USA). Vimentin (Cat# 5741), Snail (Cat# 3879), and β-actin (Cat# 3700) antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). E-cadherin (Cat# sc-71008 and sc-8426) and N-cadherin (Cat# sc-8424) antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). 6-Diazo-5-Oxo-L-Norleucine (DON, Cat# D2141) and WGA (Wheat germ agglutinin, Cat# L3892) lectin were purchased from Sigma-Aldrich (St Louis, MO, USA). Snail antibody (Cat# 13099-1-AP) was purchased from ProteinTech (Chicago, IL, USA). GFAT2 antibody (Cat# 40023) was purchased from SAB (Pearland, TX, USA). TGF-β1 neutralizing antibody (Cat# MAB1835) was purchased from R&D Systems (Minneapolis, MN, USA). AlamarBlue^® cell viability reagent (Cat# DAL1025) was purchased from Thermo Fisher (Waltham, MA, USA). Ultra-low attachment 96-well plates were purchased from Corning (Corning, NY, USA).

As described previously, astrocytes were acquired from neonatal rats (P2–3)42 (link). The mixed cells were plated onto culture flasks which were coated with poly-L-lysine, and cultured in DMEM/F12 (Gibco) containing 10% FBS (Gibco). The medium was changed every 2–3 days, and 6–10 days later, the cultures were put on shaker (2.5 g, 37 °C, 5% CO₂) overnight to remove microglia and oligodendrocyte lineage cells. The astrocytes were harvested and plated (1 × 10⁵/cm²) onto coverslips in 12-well plates for further analysis. Cells were 24 h before treatment. For IL-6 studies, astrocytes were treated with IL-6 (Sigma) at concentrations of 0, 10, 50, 100 ng/ml for further 24 h, and the level of GFAP and CCL7 were analyzed to acquire the optimum concentration of IL-6. In further studies, the optimum concentration of IL-6 was chosen to stimulate astrocytes.
Co-cultures of pure astrocytes (1.0 × 10⁵/cm²) and BMSCs or IL-1β-BMSCs (1.0 × 10⁵/cm²) were plated onto the coverslips in 24-well plates for 24 h after which IL-6 (100 ng/ml) was added. Cells were cultured for a further 24 h and the levels of GFAP and CCL7 were analyzed. For Ab blocking studies, IL-10 blocking Ab (BioLegend, San Diego, USA) or TGF-β1 neutralizing antibody (R&D Systems, Minneapolis, MN, USA) were added to the co-culture system 0.5 h before IL-6 stimulation.

Recombinant human TGF-β1 was purchased from Merck Millipore. TGF-β1 neutralizing antibody and control IgG antibody (Rabbit polyclonal IgG) were obtained from R&D systems. CAY10444, JTE-013, SB203580, sphingosine-1-phosphate (S1P), SKI-5C (CAY10621), SIS3, and W146 were obtained from Cayman Chemicals. A83-01 was purchased from Sigma-Aldrich. ALK5 inhibitor II was obtained from Enzo Life Sciences.