Llvy amc

Dissected fat body, intestine and Malpighian tubules from 2–4 fresh flies were homogenised in 25 mM Tris-HCl (pH 7.5) buffer on ice and centrifuged. Protein concentration in the supernatant was measured using Bradford reagent (Bio-Rad). 20 μg of sample was loaded onto a black 96-well plate with 25 μM of the proteasome substrate N-Succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin (LLVY-AMC; Sigma, S6510) in a 200 μl final volume. The excitation/emission wavelengths were 350/440 nm, and enzyme kinetics were recorded at 25°C in a temperature-controlled fluorimetric microplate reader (Tecan Infinite M200). Proteasome activity was quantified using 7-amino-4-methylcoumarin (AMC; Sigma, A9891) as a standard, and determined from the slope of AMC accumulation over time.

Analyses of autophagy, apoptosis, mitochondrial membrane potential and proteasome peptidase activity were performed on a BD Accuri C6 flow cytometer (BD Biosciences). Annexin V-FITC/PI apoptosis detection kit was used as described by the manufacturer (Thermo Fisher Scientific). MAP1LC3A activation was measured using the FlowCellect Autophagy LC3 Antibody-based Assay Kit (Merck Millipore). Mitochondrial inner membrane potential was measured with the JC-1 probe (Thermo Fisher Scientific) as previously described (Agier et al., 2012 (link)). The peptidase activity of proteasomes was monitored using the fluorogenic peptide succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin, LLVY-AMC (Sigma-Aldrich) (Bulteau et al., 2006 (link)).

Peptidase activity of the proteasome was assayed using a fluorogenic peptide, succinyl-Leu-Leu-Val-Tyr-7-Amido-4-Methylcoumarin (LLVY-AMC) Sigma-Aldrich (Saint Quentin Falavier, France) as previously described36 (link).