Figure 2: Effect of methotrexate (MTX) on proteoglycan synthesis and size in human vascular smooth muscles cells (VSMCs) treated with a G Protein Coupled Receptor (GPCR) (thrombin), a protein tyrosine kinase (PDGF) and a protein serine/threonine kinase (TGFβ) agonist. Serum-deprived (24 h) cells were treated with thrombin (10 u/ml), PDGF (50 ng/ml) or TGF‑β (2 ng/ml) for 24 h in the presence of MTX (10 μM) and radiolabeled with [35S]-sulfate (50 μCi/ml). A. The media containing the secreted proteoglycans was isolated and spotted onto Whattman paper and ran through CPC precipitation to quantitate proteoglycans. B. The remaining media was purified using DEAE-loaded ion exchange chromatography and subjected to SDS‑PAGE analysis to determine the apparent size of dominant proteoglycan which has previously been characterised as biglycan. The experiment was completed twice in triplicate and the data shows the combined results. Statistical analysis was performed using one-way ANOVA, ## P<0.01 (Basal vs Agonist).