Protein samples (conditioned growth media) were collected at 4 °C in PBS buffer containing Complete Protease Inhibitor Cocktail (Roche) and concentrated with Amicon Ultra-15 centrifugal filter units, with a 30kDa size exclusion (EMD Millipore). Total protein concentration of samples was determined via Bradford assay (Sigma) and equal amounts of total protein were loaded onto 12% Tris-HCl gels (Bio-Rad). Following electrophoresis (100 V for 45 minutes), proteins were transferred to Immobilon-P membranes (EMD Millipore). Blots were probed overnight at 4 °C with 1:200 rabbit anti-GLYPICAN2 (abcam, ab129526), 1:200 rabbit anti-VERSICAN (abcam ab19345), 1:1000 rabbit anti-SYNDECAN1 (Invitrogen, 36-2900), 1:1000 rabbit anti-BREVICAN (MyBioSource, MBS710876), 1:1000 mouse anti-NEUROCAN (EMD Millipore, MAB5234), 1:200 mouse anti-NG2 (abcam, ab50009), 1:50 goat anti-mouse C1q (Santa Cruz Biotechnology, sc-365301). Blots were incubated with HRP-conjugated secondary antibodies at 1:5000 for 2 hours at room temperature and developed using ECL Prime Western Blotting Detection Reagent (GE Healthcare). Visualization and imaging of blots was performed with a FluorochemQ System (ProteinSimple).
