Fig. 1. Skeletal muscle cells synthesize an extracellular heparin-binding protein that increases in concentration during differentiation. (A) Proteins soluble in PBS, TX-100 and TX-100/KCl were sequentially extracted from C₂C₁₂ myoblasts. Proteins were analyzed by SDS-PAGE and stained with Coomassie blue (left panel) or subjected to a [³⁵S]heparin ligand blot assay (right panel). (B) Heparin-solubilized proteins obtained from intact C₂C₁₂ cultures on various days of differentiation (days 0-6) were analyzed by SDS-PAGE. Gel loading was normalized on the basis of equivalent amounts of DNA per lane. The presence of the p33/30 doublet was detected by a [³⁵S]heparin ligand blot assay. (C) Proteins solubilized in TX-100/KCl obtained from myoblasts were analyzed by SDS-PAGE and stained by Coomassie blue (left panel) or analyzed in a competition [³⁵S]heparin ligand blot assay (right panel). [³⁵S]heparin was co-incubated with 2 mg/ml of unlabeled heparin (Hep), chondroitin sulfate (CS) or dermatan sulfate (DS). An equivalent amount of total protein was loaded per lane. Molecular weight markers are shown on the left. Arrows on the right indicate the migration of p33/30.