Fig. 4

Active secretion of HMGB1 following Eh contact with macrophages at early time points. a THP-1 cells were incubated with Eh for increasing times at 20:1 ratio and equal amounts of cell culture supernatant (SN) samples were loaded onto the SDS-PAGE gel to detect HMGB1. b Immunoprecipitation assay done with acetyl lysine antibody and immunoblotted with HMGB1 to detect the acetylated form of HMGB1. c THP-1 cells were pre-treated 30 min with 5 μM anacardic acid (AA) and then stimulated with Eh for 10 min to detect HMGB1 level. d Immunofluorescence analysis of HMGB1 was done in the presence or absence of AA with Eh after 10 min incubation with macrophages. Representative images from three independent experiments were shown (scale bar: 10 μm). e Quantification of the mean cytoplasmic intensity of HMGB1 shown as mean ± SEM. f HAT activity assay was performed following Eh stimulation in the absence or presence of AA. Data are representative of three independent experiments. ***P < 0.001. **P < 0.01.