Chemical cross-linking of IN1-105. The reaction was carried out in 50 mM Hepes pH 7.8. IN 1 –105 and BS3 was incubated at 25°C in 40 μL at the times and concentrations indicated. The reaction was stopped with 50 mM lysine pH 8.0. (A) Crosslinking kinetics. 56 μM of IN 1–105 were incubated with 100 μM of BS3, aliquots were removed at the times indicated. Left, SDS-PAGE (15% acrylamide) silver stained (4 μg of protein were loaded in each lane). Right, formation of crosslinked IN 1–105 dimer (percent); quantification of dimeric and monomeric band were carried out with the imageJ program. (B) Effect of BS3 in crosslinking. 56 μM IN 1–105 and BS3 (0 to 300 μM) were incubated for 35 min at 25°C, aliquots containing 4 μg were loaded on a 12% acrylamide gel. Left, SDS-PAGE (12%) Coomasie brilliant blue stained gel. Righ t, formation of crosslinked IN 1–105 dimer.