FIGURE 6.
Modulation of Pmel17 S2 cleavage by pharmacological treatment or knock-down of metalloproteases. A, HeLa cells were transfected with Pmel17-i or the QL mutant. Incubation with DAPT or coincubation with PMA for 4 h resulted in increased formation of Pmel17-CTF in case of the wild type construct but had no effect on the S2 cleavage site mutant QL. Blotting of the conditioned media using HMB45 revealed increased secretion of Pmel17 in cells transfected with Pmel17-i but not in the QL mutant. Likewise, PMA induced the formation of insoluble MαC fragments in the insoluble fraction of Pmel17-i transfected cells but failed to do so in the QL mutant. B, HeLa cells transfected with Pmel17-i were exposed to 100 μm of the inhibitor TAPI-2 for 18 h. The soluble and insoluble fractions were immunoblotted using anti-Myc or HMB45 antibodies, respectively. C, quantification of B (n = 2 ± S.E., performed in duplicate; ***, p < 0.001, Student's t test). D, HeLa cells were cotransfected with Pmel17-i and control siRNAs or siRNAs targeting the metalloproteases ADAM10 and ADAM17. Cell lysates were immunoblotted using Myc antibody for Pmel17. Pellet fractions were SDS-extracted and analyzed using antibody HMB45 (n = 3 ± S.E., performed in duplicate; **, p < 0.01; ***, p > 0.001, one-way analysis of variance, Tukey post hoc test). E, the effectiveness of the siRNA knock-down was verified by immunoblotting the lysates for expression of ADAM10 and ADAM17. Knock-down of ADAM10 resulted in a decrease of the proform (P) and the mature form (M). The latter was used for quantification (n = 3 ± S.E., performed in duplicate; **, p < 0.01, Student's t test).