Biglycan Stimulation of Endothelial Cells

ECs were grown to subconfluence and culture medium was replaced with fresh EGM-2 MV. After 18–24 h, CM was collected and passed through a 0.22-μm filter (Merck Millipore) and subsequently concentrated approximately 120-fold using Amicon Ultra-15 30 K centrifugal filter units and Amicon Ultra 0.5-mL 30 K centrifugal filters (Merck Millipore). LM-tumour cells were preincubated with anti-TLR2 and/or TLR4 antibodies (Biolegend) for 90 min, or with U0126 or BAY11-7082 for 60 min. Cells were lysed after stimulation of biglycan for 30 min (ERK) or 60 min (NF-κB). Equal amounts of total protein were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PDVF) membranes. Equal loading and transfer were confirmed by MemCode Reversible Protein Stain Kit (Thermo Scientific) for detection of biglycan in the CM of ECs. Western blotting was performed using antibodies listed in Supplementary Table S1 and an HRP-conjugated secondary antibody, as previously described10 (link)12 (link).