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Figure 5 | Respiratory Research

Figure 5

From: Extracellular Hsp72, an endogenous DAMP, is released by virally infected airway epithelial cells and activates neutrophils via Toll-like receptor (TLR)-4

Figure 5

Exogenous Hsp72 activates NF-κB in HL-60 cells. A. Differentiated HL-60 cells were pretreated with isohelenin (30 μM) or DMSO for 1 h prior to treatment with Hsp72 (100 ng/ml). Supernatants were harvested, clarified and run on IL-8 ELISA. Data represent means ± SEM for 3 experiments and differences pinpointed by ANOVA (compared to control *p < 0.001). B. HL-60 cells were treated with Hsp72 (100 ng/ml) for times as indicated (0–240 minutes). Cell lysate was harvested, run on a Western blot and probed for IκBα. Shown is a representative Western blot. This experiment was performed 4 times. C. Quantification of the IκBα western blots. Data are expressed as percentage of IκBα remaining compared to untreated sample for each experiment (mean ± SEM for 4 experiments *p = 0.014, **p = 0.005). D. Nuclear extracts from cells treated with or without Hsp72 (100 ng/ml) were incubated with an oligonucleotide NF-κB consensus sequence and EMSA was performed. Antibodies against p65RelA or p50 NF-κB were added to selected samples. Cold NF-κB probe (5× excess) or cold AP-1 probe (5× excess) was added to a sample to show specificity. These data are representative of two separate experiments.

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