Figure 2.

Increasing HAI-2 expression only results in modest suppression of active matriptase shedding. (A) Daudi cells were engineered to express HAI-2 in a doxycycline-regulated manner (Teton HAI-2). Teton HAI-2 cells were treated with increasing amounts of doxycycline. Lysates from an equal number of cells were then analyzed by Western blot for HAI-2 (left panel) and total matriptase (right panel) levels. (B–D) Teton HAI-2 cells were treated with increasing doxycycline concentrations to induce the expression of increasing levels of HAI-2, after which an equal number of these cells were induced to activate matriptase by acid buffer exposure. The cell lysate and the conditioned buffer (shed fraction) were collected and adjusted to the same volume. An equal volume of the cell lysates (B) and the shed fraction (C) were analyzed by Western blot for HAI-2 species (B), total matriptase (B) and (C), and activated matriptase (B and C), and by gelatin zymography for matriptase gelatinolytic activity (C), and by the amidolytic activity assay using a fluorogenic peptide substrate (D). The HAI-2 and matriptase species, including cell-associated HAI-2 (band a), cell-associated matriptase (band b), matriptase-HAI-2-protease X complex (band c), matriptase-HAI-2 complex (band d), shed matriptase (band e), shed matriptase fragment (band f), shed active matriptase (band g), and shed active matriptase fragment (band h) are indicated. These experiments to evaluate the impact of HAI-2 expression on the shedding of active matriptase were performed at least three times, and representative data are presented. ns: non-significant difference; ∗∗: P < 0.01.