Bml p802 0005

Proteasome activity was evaluated using yeast proteasome (TUM Groll group). The inhibitory potency of the extracts against yeast proteasome was assessed by using the Fluorescence Intensity Assay and Suc-Leu-Leu-Val-Tyr-AMC as substrate (Enzo Life Sciences, BML-P802-0005). The assay was carried out at room temperature (22 °C) using Corning 4514 black low volume 384 well plates. All measurements were performed in singlicate and the final DMSO concentration was 3.3%. The assay buffer contained 100 mM Tris pH 7.5 and 1 mM MgCl2. First, 9.5 µL protein dilution (final concentration 9 nM) were mixed with 0.5 µL of either one of three probe dilutions (1, 0.1 or 0.01 mg/mL >> assay end conc. 0.033, 0.0033 or 0.00033 mg/mL) and preincubated for 90 min. Then 5 µL substrate mix were added (final concentration 5 µM) and incubated for 60 min. The assay plates were measured in fluorescence mode on a Tecan M1000 plate reader (ex 380 nm, em 460 nm); the IC50 was calculated using XLfit. ONX-0914, which inhibits yeast proteasome, was used as positive control in order to assess the functionality of the assay.

HeLa cells were seeded in 6-well plates and the assay was performed in triplicates. First, the cells were washed twice with PBS, then collected in 1.5 ml Eppendorf tubes and centrifuged at 2,000 r.p.m. for 5 min at 4 °C. The cellular pellet was resuspended in 100 μl 50 mM Tris, pH 7.4, I mM DTT and sonicated twice for 10 s. The cellular debris was removed by centrifugation (13,000 r.p.m., 10 min, 4 °C), then the supernatant was collected in a separate tube. For measuring the chymotrypsin-like activity of the proteasome, 25 μg proteins were used per reaction together with 100 μM of Suc-LLVY-ACM (Enzo, BML-P802-0005). The activity was measured for 1 h at 37 °C at 5 min interval using a plate reader with Ex. 380 nm and Em. 460 nm.

The forebrain and spinal cord proteins were extracted freshly. This assay was carried out in 96 well plates with 50 μg of the extracts lysate and fluorescently labeled substrate succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin (Suc-LLVY-AMC; final conc. Was 100 μM; BML-P802–0005, Enzo Life Sciences, NY) in assay buffer [20 mM Tris-HCl (pH = 7.4), 5 mM MgCl2, 5 mM DTT and 2 mM ATP]. The kinetics of the Suc-LLVY-AMC cleavage was measured spectrofluorometrically at 5 min intervals for 1 h under 37 °C by using an EnSpire Multiplate Reader (Ex/Em = 360 nm/460 nm; PerkinElmer). Negative control assay was conducted in the presence of the specific proteasomal inhibitors MG-132 (474,790, Calbiochem) and Lactacystin (70,988, Cayman).