Fig. 1. HMGB1 is multiply acetylated in thymus and activated monocytes. (A) HMGB1 purified from calf thymus gives rise on a monodimensional gel (SDS–PAGE, Coomassie stain, right panel) to two bands (arrows). The minor band contains ADP-ribosylated HMGB1. Fifty micrograms of the same sample of HMGB1 was subjected to 2D gel electrophoresis (silver stain, left panel); 2D Protein Marker from Bio-Rad was loaded together with HMGB1, generating a matrix of spots at the molecular weights listed on the right. (B) Total mouse thymus contains many isoforms of HMGB1. About 300 µg of protein from a thymus total extract (from a 17-day-old mouse embryo) were loaded on twin 2D gels; the gel shown at the top was silver stained, while the one on the bottom was blotted and assayed with anti-HMGB1 antibody. (C) LPS-activated human monocytes hyperacetylate HMGB1 and accumulate it in cytoplasmic vesicles. Monocytes purified from peripheral blood were cultured overnight, with or without LPS. Aliquots of activated and control monocytes were then fixed and immunostained with anti-HMGB1 antibody (red). HMGB1 is nuclear in unstimulated monocytes, as opposed to nuclear plus vesicular in LPS-activated monocytes. Bar represents 7 µm. Aliquots of untreated and LPS-activated monocytes were freeze-thawed, and about 400 µg of total protein extract was loaded onto 2D gels, blotted and immunodetected with anti-HMGB1. Note the major additional HMGB1 spot in activated monocytes.