Heparin sepharose beads

Plasma, CSF or h295-R cell-conditioned media were enriched for CCN3 using heparin-sepharose beads (GE Healthcare, #17-0998-01) as previously described [35 (link)]. In short, up to 4 ml (equivalent to at least 20 ng of CCN3) was added to 20 μl 50% heparin-sepharose slurry and incubated on a rotator at 4 °C overnight. In total, 25 μl of PBS-washed CCN3-bound heparin-sepharose beads and 5 μl of 6× reducing loading dye were boiled for 10 min prior to loading on a reducing 15% SDS-PAGE. Following transfer onto PVDF membrane (Millipore) and blocking (3% BSA in PBS/1% Tween) for 1 h at room temperature, protein was probed using polyclonal goat anti-human CCN3 antibody (0.1 mg/ml; cat. no. AF1640, R&D Systems) overnight at 4 °C and secondary rabbit anti-goat HRP (1:2000; #61-1620) for 1.5 h at room temperature. Bands were detected by chemiluminescence using Pierce™ ECL Western Blotting Substrate (Thermo Fisher, #32106) and imaged using a G:BOX detection system (Syngene).

Recombinant human VEGF‐A was from R&D Systems (Minneapolis, MN) (cat#293‐VE) or PreproTech (Rocky Hill, NJ), (cat#100‐20); Heparin‐Sepharose beads from GE Healthcare (Piscataway, NJ); PMA (cat# P8139) and cycloheximide (CHX) (cat# C4859) from Sigma (St. Louis, MO); pharmacological inhibitors, see Table 1, from EMD Calbiochem (San Diego, CA) or SelleckChem (Houston, TX). All general reagents were from Thermo‐Fisher (Pittsburgh, PA), unless otherwise stated.