St1120

For Western Blot analysis of IDE and β-actin protein levels, samples were prepared as described above, adjusted to equal protein amounts and loaded on 10–20% tris-tricine-gradient gels (Anamed Elektrophorese, Groß-Bieberau/Rodau, Germany). For measuring total Aβ degradation, the cell culture supernatant containing remaining human Aβ40 was also separated in tris-tricine-gradient gels. After transferring proteins onto nitrocellulose membranes, IDE, β-actin and human Aβ40 were detected with the primary antibodies ST1120 (1:2000), A5441 (1:5000) and W02 (1 μg/mL), all purchased from Merck, respectively. HRP-coupled antibodies W401 (anti-rabbit, 1:5000) (Promega, Mannheim, Germany) and P0260 (anti-mouse, 1:5000) (Dako, Hamburg, Germany) were utilized as secondary antibodies. Signal detection was performed with the enhanced chemiluminescence (ECL-) method (Perkin Elmer, Rodgau-Jügesheim, Germany) and densitometrical quantification of band intensity was carried out with Image Gauge software (Version 3.45) (Fujifilm). Total proteins were detected using Ponceau S staining as described in [52 (link)] before immunodetection.

IDE enzyme activity was analyzed as described in^{70 (link)} with minor modifications. Anti-IDE antibody ST1120 (Merck) was diluted in 1xPBS (5 µg/ml) and coated on a Nunc MaxiSorp 96-well plate for 24 hours at room temperature. After washing the plate five times with 1x PBS containing 0.5% Tween-20, blocking was performed by incubating 10% fatty acid free bovine serum albumin in 1x PBS for two hours at room temperature. Afterwards, plate was washed three times and lysates were incubated for one hour at 20 °C and 150 rpm, followed by five wash steps. After a pre-incubation with assay buffer (50 mM Tris-HCl pH 7.4, 1 M NaCl, 10 µM MgCl₂, β-secretase inhibitor II (565749, Merck), γ-secretase inhibitor IV (565761, Merck), complete protease-inhibitor without EDTA) for 15 minutes at room temperature, the fluorogenic peptide substrate Mca-RPPGFSAFK(Dnp)-OH (10 µM) was added and fluorescent was measured using a Safire² Fluorometer (Tecan, Crailsheim, Germany) at an excitation wavelength of 320 ± 10 nm, and an emission wavelength of 405 ± 10 nm.