Figure 9
Affinity chromatography of 35 SO 4 -labeled proteoglycans on III1-C Sepharose. Panel A. RASMCs were labeled with 35SO4, which preferentially labels the glycosaminoglycan chains of proteoglycans. Cells were lysed in NP40 buffer and lysates were applied to either III1-C Sepharose (C lanes) or III 11-C Sepharose (11C lanes) columns. The flow through fractions were collected (Flow Thru lanes), the columns were washed, and the bound material was eluted by first applying 8 M urea elution buffer (Urea lanes), washing the columns with PBS, then collecting the Sepharose beads and boiling them in SDS-PAGE sample buffer (SDS lanes). The lane labeled St shows a sample of the starting material. All samples were separated on SDS-PAGE gels then detected by phosphorimager analysis. Numbers to the left of the panel indicate the migration of molecular mass markers (kDa). Panel B. Samples of the starting material were treated with either no enzyme ((-)), or with 0.03 u/ml chondroitinase ABC (C'ase), or 0.03 u/ml heparitinase (H'ase), then analyzed by SDS-PAGE and phosphorimager. The experiment was performed twice with similar results both times.