Soluble Heparan Sulfate Fragments Generated by Heparanase Trigger the Release of Pro-Inflammatory Cytokines through TLR-4
Figure 3
Proteinase-K treated HPSE results in a reduced cytokine response in PBMCs.
HPSE (1 µg) was treated with proteinase-K agarose beads (80 µg/ml) for 30 min at 37°C before removal of proteinase-K agarose beads and SDS-PAGE analysis. (A) Western blot analysis for HPSE confirms that proteinase-K treatment dramatically reduced intact HPSE levels in the sample. (B) HPSE activity assay on identically treated samples of either HPSE alone, proteinase K alone, or HPSE treated with proteinase K. Data represent the mean ±SEM (n = 3). (C) Expression of cytokines after stimulation with proteinase-K treated HPSE in IL-1β, IL-6, IL-10, IL-8 and TNF in PBMCs isolated from human whole blood. Data represent the mean ±SEM of triplicate samples, results are representative of three independent experiments. *p = <0.05, **p = <0.001; unpaired, two-tailed Student's t-test.