HMGB1 is released into the cytoplasm and supernatant of α-toxin treated cells.

<p>(A–B) Intracellular translocation of HMGB1 was visualized using fluorescence microscopy and Western blotting. (A) Cell nuclei were stained with DAPI and HMGB1 localized using anti-HMGB1 antibodies and an Alexa Fluor-488 conjugated secondary antibody. Images are representative of the results from five separate experiments. (B) Western blot analysis of HMGB1 in cytoplasmic (Cyt) and nuclear (Nuc) fractions. (C) Extracellular translocation of HMGB1 was confirmed by Western blot analysis of whole cell lysates (WCL) and culture supernatants (Sup) of α-toxin treated cells compared to untreated control cells. Brackets indicate the concentration of α-toxin in µg/ml.