FGF2 monomers and dimers after cross-linking and variant forms of FGF2 after proteolytic degradation. (A) FGF2 (1.5 μg) in Tris-HCl (pH 7.5) was incubated with 1 mM ATP and 10 μg/mL heparin and 20 μg/mL protamine at 37°C. After 15 min, 1 mM BS3-crosslinker was added and the samples were incubated for 1 h at 20°C. Reactions were stopped by addition of SDS-loading dye and boiling. The proteins were separated by 15% SDS-PAGE and visualized by silver staining. (B) Two μg FGF2 (K134A) in Tris-HCl (pH 7.5), were incubated with ATP (100 μM) or without ATP at 37°C. After 15 min FGF2 and FGF2 (K134A) were incubated with 200 ng NE at 37°C for 2 h. The reactions were stopped by addition of SDS-loading dye and boiling. The samples were separated by SDS-PAGE and protein was visualized by silver staining.