Figure 3

Concentration-dependent binding curves of esRAGE and D₆-esRAGE to HMGB1. His-tagged HMGB1 was incubated and immobilized to Nickel affinity gel suspension in PBS containing 4 mmol/L imidazole, and esRAGE or D₆-esRAGE was then added to the suspension at a final concentration of 4, 8 and 16 µg/mL. The mixture was agitated at 37°C for 30 min to allow the binding of esRAGE and D₆-esRAGE to HMGB1 immobilized in Nickel affinity gel. After washing the Nickel affinity gel with PBS containing 4 mmol/L imidazole, the bound esRAGE and D₆-esRAGE was eluted from the gel complex by incubating in PBS containing 250 mmol/L imidazole. The eluted esRAGE and D₆-esRAGE was determined by Western blot analysis and the relative intensities of immunoreactive blots were calculated. The concentration-dependent binding curves were comparable between esRAGE and D₆-esRAGE. Each point with a bar represents the mean ± standard deviation (SD) of three experiments.