Figure 4.

Assay of TACE-like activity using the TACE fluorescent substrate Fluorogenic Peptide Substrate III. A, Serial dilutions of protein extracts from hypothalamic astrocytes incubated with a single concentration (10 μg) of fluorescent substrate. The reactions were performed at 37°C, and the fluorescence was determined at 10 min intervals. B, Linear correlation between the intensity of the fluorescent reaction, determined after 2 h of incubation, and the amount of protein added to the reaction. C, The specificity of the reaction was determined by incubating the protein extracts with increasing doses of TAPI-2 (a selective TACE inhibitor). The TACE activity measured after 2 h of incubation was dose dependently inhibited by TAPI-2, with a maximum of 65% inhibition at a 50 μm dose (D). **p < 0.01 and ***p < 0.001 versus control group (n = 7–8 independent observations per group). RFU, Relative fluorescence units.