B1–B3, but not B4 beads bind to HMGB1.

A) Depletion curve of HMGB1 binding to various DNA beads. Recombinant HMGB1 (2 µg per reaction) was mixed with a range of concentrations of different types of DNA beads as indicated and incubated at room temperature for two hours. The mixture was then centrifuged at 2,000 rpm for five minutes to precipitate the beads. Beads were then washed five times with PBS. Both supernatants and elute from beads were subjected to western blot analysis probed with anti-HMGB1 antibody. B) Saturation curve of HMGB1 binding to various DNA beads. Fixed amounts (20 µl drained beads) of B1–B3 or control beads were incubated with increasing amounts of HMGB1 (in 50 µl volume) at concentrations indicated for two hours at room temperature with rotation. The mixture was then centrifuged at 2,000 rpm for five minutes to precipitate the beads. The supernatants were aspirated, both supernatants and elute from beads were subjected to western blot for HMGB1 measurement using monoclonal anti-HMGB1 antibodies. The binding of 1 µg HMGB1 requires about 0.4 ng (B1 and beads) or 2.8 ng (B3) in beads, respectively. C) Time-course of HMGB1 binding to various DNA beads. B1–B3 beads (20 µl) were incubated with 500 ng of HMGB1 (50 µl total volume) at room temperature for the time periods indicated. HMGB1 bound to the beads was revealed by western blot analysis. Data are representative from 3–4 separate experiments.