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. 2014 Jan 21;9(1):e87014. doi: 10.1371/journal.pone.0087014

Figure 5. Single knock-down of ADAM9, ADAM10, ADAM17 and MMP9 does not affect 5-HT4d receptor-stimulated APP shedding.

Figure 5

(A) SH-SY5Y cells, transfected with pEAK12-AP-APP and pcDNA3.1-5-HT4d, were treated with 1 µM prucalopride or 5-HT (5-HT4 receptor agonists) in the absence or presence of 80 µM GM6001 (metalloproteinases inhibitor) and secretion of sAPPα was analyzed via measuring SEAP. (B) SEAP levels were measured in supernatants of SH-SY5Y cells, co-transfected with pEAK12-AP-APP, pcDNA3.1-5-HT4d and 3 nM siRNA for knock-down of ADAM9 (A9), ADAM10 (A10), ADAM17 (A17) and MMP9 and treated with 1 µM prucalopride. (C) Cell lysates of (B) were analyzed for protein expression of ADAM9, 10, 17 and MMP9 by western blotting. The ADAM10 and ADAM17 immature precursor proteins are indicated by an x, whereas the mature catalytically active forms are indicated by an xx for ADAM9, 17 and MMP9. The immature ADAM9 and the mature ADAM10 proteins were not visible. (D) Quantification of experiments in (C). Values shown are mean ± SEM of 6 individual wells and were normalized to vehicle control. * P<0.05, ** P<0.01, *** P<0.001, one-way ANOVA with Tukey-Kramer's post-hoc test.