Figure 1

The expression of HMGB1 on microparticles from RAW 264.7 macrophage cells. RAW 264.7 cells were cultured for 20 h at 37°C, 5% CO₂ with etoposide (ET; 30 µg/mL), lipopolysaccharide (LPS; 50 µg/mL) or staurosporine (STS; 1 µmol/L) or cultured without treatment (UN). Cells were removed by low speed centrifugation and MPs were purified by two rounds of centrifugation at 100,000g for 25 min. The concentrated MP samples were subjected to SDS gel electrophoresis under reducing conditions using equal volumes of sedimented particles. The HMGB1 was detected by Western blotting with a rabbit anti-mouse HMGB1 antibody preparation, using as controls supernatants from Jurkat cells that were cultured without treatment (UN) or heated to induce death (Heat). After incubating with an anti-rabbit HRP-conjugated antibody, the membrane was incubated with Super-Signal West Femto maximum sensitivity substrate and the resulting signal detected.