Fig. 4

Hsp70 forms a complex with another DAMP, HMGB1. (A) Venn diagram of Hsp70-binding protein precipitated with anti-Hsp70 antibodies from untreated A549 cells and treated with etoposide. (B) Interactome of Hsp70 released from untreated A549 cells (left panel) divided to six clusters; in right panel: interactome of Hsp70 released from etoposide-treated A549 cells. Among Hsp70-binding proteins found in Eto-CM but not in CM from untreated cells, HMGB1 was found. (C) The retrospective analysis of overall survival and disease-free survival in lung and colon cancer patients carrying tumors with high and low levels of HMGB1. (D) FRET analysis of purified Hsp70-HMGB1 interaction. Representative emission spectra detected at 516–600 nm, excitation at 488 nm. The HMGB1-555 concentration is indicated in µg/ml in brackets. The Hsp70-488 concentration was 50 µg/ml. (E) Scheme of Protein-Protein Interaction assay (PPI). (F) results of PPI using pure HMGB1 and Hsp70. Antibody to HMGB1 (capture AB) was placed on the bottom of 96-well plates and then HMGB1 at a concentration of 1 or 10 µg/ml was added. After blocking, Hsp70 at concentrations of 0.1, 1.0 or 10 µg/ml was applied. Hsp70 in complex was identified with biotinylated anti-Hsp70 antibody (see (E)). (G) Western blotting of ATP or IP eluates (P) and unbound fraction (S) from Eto-CM or Oxa-CM from A549 cells. The membranes were probed with anti-HMGB1 and anti-Hsp70 antibodies. (H) Data of pull-down assay with biotinylated Hsp70 or biotinylated HMGB1 in Eto-CM and Oxa-CM of A549 cells