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. 2015 Nov 2;5:15971. doi: 10.1038/srep15971

Figure 2. LPS promotes HMGB1 release via its dissociation from SIRT1.

Figure 2

(A–C) HEK293T cells co-transfected with Flag-HMGB1 and/or Myc-SIRT1 for 48 h were incubated with LPS (100 ng/ml), Poly (I:C) (50 μg/ml), IFN-γ (40 ng/ml), or TNF-α (20 ng/ml) for 3 h, and then whole-cell lysates were immunoprecipitated with an anti-Flag (A,C) or anti-Myc (B) antibody and analyzed by Western blotting. (D,E) HEK293T cells expressing Flag-HMGB1 were incubated with or without the indicated stimuli for 3 h. Whole-cell lysates were incubated with recombinant GST or GST-SIRT1 fusion protein immobilized to glutathione-Sepharose 4B beads for 20 h, and then pulled down or immunoprecipitated. GST and GST-fused proteins were stained with Ponceau S. (F) RAW 264.7 cells co-transfected with Flag-HMGB1 and Myc-SIRT1 for 48 h were incubated with or without LPS (100 ng/ml) for 6 h (for interaction) or 24 h (for HMGB1 release). Whole-cell lysates were immunoprecipitated with an anti-HMGB1 antibody to determine the interaction with SIRT1. To detect released HMGB1, equal volumes of conditioned media were analyzed by Western blotting.