Figure 10

Affinity chromatography HSPGs on III1-C Sepharose. RASMCs were collected by trypsinization and lysed in NP40 buffer. The lysate was then treated either without (-) or with (+) 0.1 u/ml heparitinase for 1 hr at 37°C. Heparitinase treated samples were then applied to either III1-C Sepharose (lanes marked C) or III 11-C Sepharose (lanes marked 11C). One sample was applied to a III1-C Sepharose column in the presence of 0.5 mg/ml heparin (lanes marked C•H). The flow through fractions were collected (FT lanes), the columns were washed extensively, then the Sepharose beads were collected and boiled in SDS-PAGE sample buffer (Bound lanes). Samples were analyzed by immunoblotting with the 3G10 antibody. Antibody 3G10 recognizes the uronate stubs that remain associated with core proteins after heparitinase digestion. Thus, after heparitinase digestion 3G10 shows the sizes of HSPG core proteins. Note that the major HSPGs have core protein sizes of 46 kDa and 70 kDa, and both of these HSPGs bind to the III1-C column but not the III 11-C column. The experiment was performed twice with similar results both times.