Figure 1.

HMGB1 is associated with nucleosomes spontaneously released during secondary necrosis. For induction of secondary necrosis (SN), Jurkat cells were treated with 2 μM staurosporine for 48 h. Primary necrosis was induced by heating cells at 56°C for 30 min followed by incubation for an additional hour at 37°C. Supernatants from apoptotic (top) and necrotic (bottom) Jurkat cells were collected and subjected to immune precipitations with anti-HMGB1, anti-dsDNA, antihistone H3, antihistone H2B, and antihistones H2A/H4 as well as with appropriate isotype control antibodies. Immune-precipitated material was then fractionated by SDS-PAGE on 12% polyacrylamide gel. Western blot analysis was performed using polyclonal anti-HMGB1 antibodies. WB, Western blotting; IP, immunoprecipitation. These experiments have been performed four times with virtually identical results.