FIGURE 8.

Meprin β shedding in primary renal tubular epithelial cells (RTEC) treated with inhibitors TAPI-1 or GI254023X in the presence or absence of PMA and ionomycin. Primary renal tubular epithelial cells were treated with 50 nm PMA or 2.5 μm ionomycin or vehicle (DMSO) for 30 min prior to the addition of 25 μm TAPI-1, 2 μm GI254023X, or vehicle. After 2 h, medium supernatants were collected and centrifuged at 500 × g for 5 min to remove cells, and the supernatants were centrifuged at 100,000 × g for 30 min to remove cellular debris. Aliquots of equal protein content were analyzed by Western blots for the presence of meprin β. Recombinant human promeprin β lacking its membrane anchor purified from HEK293 cells described earlier (21) is shown as a reference (left panel). Albumin detected by Ponceau S stain is shown as a loading control. Western blot intensities were evaluated by densitometry normalized to those observed in medium supernatants of the corresponding control (right panel). Numbers represent the average of 5 independent experiments. Error bars represent S.E., n = 5. * p < 0.05, ** p < 0.01, *** p < 0.001.