Figure 2.

Specific ADAM10 inhibitors block the postlysis degradation of ADAM10. A) HEK293 cells were lysed with lysis buffer that contained GI254023X (5 µM), 1,10-phenanthroline (Phe.; 10 mM), TAPI-1 (50 µM), MN8 (5 µM), LT4 (5 µM), or DMSO as control. For the detection of ADAM17, lysates were subjected to an overnight ConA pull down. B) SH-SY5Y cells were lysed with lysis buffer that contained GI254023X (5 µM) or DMSO as control. C) Primary murine neurons (E16.5) were lysed with lysis buffer that contained GI254023X (5 µM) or DMSO as control. D) HEK293 cells were lysed with lysis buffer that contained GI254023X (5 µM) or DMSO as control. Cells were kept on ice for 60 min. After the indicated time points (0, 5, 10, 30, or 60 min), GI254023X (5 µM) was added to lysates, which previously did not contain GI254023X, to stop the reaction. Shown are representative Western blots. For the detection of ADAM10, C-terminal antibody was used. E) Quantification of mean proADAM10 and mADAM10 levels (n = 6) relative to those at the starting time point. Shown are means ± sem. Relative log₂ transformed values using 2-sided Student’s t test with Welch’s correction and post hoc Bonferroni’s correction. *P < 0.05, **P < 0.01, ***P < 0.001.