Figure 2: ADAM17 inhibitors abrogate PS exposure.
From: Phosphatidylserine exposure is required for ADAM17 sheddase function
HaCaT keratinocytes were stimulated with melittin (Mel, 1 μM), IO (1 μM), PMA (300 ng ml−1) or FGF7 (100 ng ml−1) in the presence or absence of P2 receptor inhibitor suramin (Sur, 50 μM), EGTA (10 mM), PKC inhibitor staurosporine (Stauro, 1 μM) or Src-kinase inhibitor (PP2, 10 μM). (a) pERK1/2 analyses were undertaken as readout for ADAM17-dependent EGFR transactivation 15 min after stimulation. Representative western blottings show abrogation of ERK1/2 activation in the presence of the inhibitors. Total ERK1/2 immunoblots are included as control. (b) The supernatant was analysed for soluble TGF-α by enzyme-linked immunosorbent assay (ELISA) 30 min after stimulation. All stimuli significantly increased soluble TGF-α shedding (n=3–6; ±s.e.m.; *P<0.05). Stimulation inhibitors, broad spectrum metalloprotease inhibitor MM (10 μM), ADAM17/10 inhibitor (GW, 2 μM) but not ADAM10 inhibitor GI (2 μM) significantly reduced TGF-α shedding (n=3–6; ±s.e.m.; #P<0.05). NS, nonsignificant. (c) Cells were stained with Annexin V–FITC 15 min after stimulation in the presence or absence of the respective inhibitors. Representative images are shown. The mean fluorescence area was quantified for statistical analysis. *A significant increase compared with untreated controls (n=3; ±s.e.m; *P<0.05). #Significant decrease compared with stimulated cells. Data were analysed by one-way analysis of variance and Bonferroni multiple comparison post hoc test. Scale bars, 10 μm.
