Enzymatically active matriptase is shed to extracellular milieu.

<p><i>A.</i> MCF7 cells were induced to activate matriptase by a pH 6.0 buffer followed by neutralization to pH 8.0. The mixture of the cells and the buffer (Combine), the pelleted cells after centrifugation (Cell), and the buffer supernatant (shed fraction) alone (Shed) were analyzed for tryptic activity by cleavage of a synthetic fluorescent substrate with Arg as P1 site. RFU stands for relative fluorescent unit. B. The shed fractions collected from MCF7 cells post induction of matriptase activation (Act.) and the non-activation control (Non-act.) were subjected to immunodepletion with activated matriptase mAb M69 beads. The shed fractions (L, lanes 1 and 4), the depleted fractions (D, lanes 2 and 5) and the eluted fractions (E, lane 3 and 6) were analyzed for matriptase species by Western blot using the mAb M24. <i>C.</i> and <i>D.</i> The shed fraction collected from pH 6 buffer-exposed MCF7 cells was immunodepleted using M69 beads. The shed fraction (L, lane 1) and the immunodepleted fraction (D, lane 2) were analyzed for the tryptic activity (panel <i>C</i>) and gelatinolytic activity by gelatin zymography (panel <i>D</i>, Gel. Zym.). RFU stands for relative fluorescent units; A–L for activated-loading; A–D for activated-depleted.