Purification of human recombinant HMGB1 and comparison with tissue-isolated HMGB1.

(A) Samples of bacteria cell lysate (lane 1) and purified rhHMGB1 (lane 2) resolved under denaturing conditions in a 4% to 20% SDS-PAGE and stained with Coomassie Blue. (B) Recombinant human HMGB1 (lane 1) and HMGB1 isolated from A549 cells (lane 2) were resolved in 4% to 20% SDS-PAGE, blotted onto nitrocellulose membranes, and incubated with the following rabbit polyclonal antibodies: (i) antisynthetic peptide gkgdpkkprgk (N-terminal sequence of human HMGB1, amino acids 2-13), (ii) antisynthetic peptide: kaekskkkkee (C-terminal sequence, amino acids 177-188), (iii) antisynthetic peptide kfkdpnapkrppsa (middle sequence, amino acids 88-101), and (iv) anti-HMGB1 (whole molecule). The antibodies were detected with horseradish-peroxidase antirabbit IgG.