(A) Binding of ³⁵S-labeled HS to cytokines and matrix components. Binding was examined by affinity coelectrophoresis, as described in Materials and Methods. Proteins cast in the lanes were 500 nmol/L ovalbumin (OVA), 25 nmol/L bFGF, 275 nmol/L FN, 100 nmol/L TSP, 500 nmol/L MIP-1, 275 nmol/L IL-3, and PF4 (at the indicated concentrations). HS (40,000 to 50,000 dpm³⁵S per gel) from supportive and nonsupportive cells were electrophoresed into the protein-containing gels. The right-side panel in the autoradiograph for the supportive HS shows lack of binding to OVA and FN. ³⁵S radioactivity was detected by autoradiography. The arrows indicate the extent of migration of unbound HS. The arrowheads indicate retardation of migration of HS due to binding to proteins in the gel. (B) Binding of ¹²⁵I-HS to TSP. ¹²⁵I-labeled HS (10,000 cpm ¹²⁵I per lane) from the two cells was electrophoresed through ovalbumin (OVA; 500 nmol/L) or the indicated concentrations of TSP. The migration of a component of the supportive HS was retarded by binding to TSP in a dose-dependent manner, compared with migration through ovalbumin. No binding was seen for the nonsupportive HS.