Figure 2

Proteinase K-agarose digestion ablates HSP70 activity. (A) 200 μg rhHSP70 was incubated with digestion buffer (lane 1), Proteinase K-agarose beads (lane 2), or control agarose beads (lane 3) for 3 hr at 37°C, as described in Methods. Protein integrity and endotoxin content were determined by silver staining and the LAL assay, respectively. (B) 100 pg of ultrapure LPS, which is an approximation of the endotoxin content in 200 μg of rhHSP70 (see Results and Discussion), was incubated with Proteinase K-agarose beads or control agarose beads, under the conditions described in A. The ultrapure LPS treated with Proteinase K-agarose beads (PK) or control agarose beads (CTRL) were then used to stimulate primary human macrophages for 16 hr at 100 pg/mL LPS final concentration in each culture. Cell culture supernatants were collected and analyzed for TNF-α. (C) The HSP70 preparations in A were used to stimulate primary human macrophages for 4 hr at 10 μg/mL final concentration. Cell culture supernatants were collected and analyzed for TNF-α. Endotoxin levels shown at bottom are the calculated amounts in the macrophage cultures based on the LAL determinations in A. Untreated macrophage cultures produced ≤ 100 pg/mL of TNF-α under these conditions. Results shown are representative of two independent digestion and stimulation experiments. ***p < 0.0005.