Suc llvy amc

Two million cells were lysed in 300 μL proteasome lysis buffer (50 mM HEPES pH 7.8, 10 mM NaCl, 1.5 mM MgCl₂, 1 mM EDTA, 1 mM EGTA, 250 mM sucrose and 5 mM DTT in PBS without Ca²⁺ or Mg²⁺). Cell lysates were sonicated 3 s using the microtip output set on ∼3 and centrifuged at 16,000 RCF for 10 min at 4°C. The total proteins were quantified from the supernatant. Equal amounts of total protein were diluted in proteasome lysis buffer supplemented with 2 mM ATP (Sigma-Aldrich, St. Louis, MO, USA) and incubated with 100 μM of proteasome substrate reporters. The following chymotrypsin-, trypsin- and caspase-like substrate reporters were used: Suc-LLVY-AMC from Enzo Life Sciences (#BML-P802-0005, Farmingdale, NY, USA); Z-ARR-AMC from Calbiochem (#CAS 90468-18-1, San Diego, CA, USA); and Z-LLE-AMC from AdipoGen (#CAS 348086-666-8, Epalinges, Switzerland). Fluorescence, A₃₆₀ex/A₄₆₀em, was measured during 60 min at 37°C with SpectraMax i3 (San Jose, CA, USA). AMC (#K245-100-4, BioVision Milpitas, CA, USA) was used as technical positive control. Purified human proteasome complexes (#E−365-025, Bio-Techne, MN, USA) were used for the cell-free proteasome activity assays.

The AMC conjugated peptide, Suc-LLVY-AMC (Enzo; BML-P802-9090) was used to assess the chymotrypsin-like proteasome activity. The purified human proteasome (0.1 μM) or tissue homogenate (5 μg per reaction) was diluted in assay buffer (50 mM HEPES, 20 mM KCl, 5 mM MgCl₂, 1 mM DTT, 10 mM ATP (pH 7.4), 0.03% SDS). To control for the non-proteasomal-mediated cleavage of substrates, 0.5 μM epoxomicin, MG132, MV151, or MV152 was added to the reaction mixture for 10 min, followed by the addition of 75 μM Suc-LLVY-AMC substrate. The activity was monitored by continuous fluorescent measurement of the released AMC at 30 °C at 1.5 to 2 min intervals for 10 min or 1 h using a microplate fluorescence reader (Infinite 200 PRO; λ_ex: 360, λ_em: 460).

RPMI8226 cells were collected in proteasome activity assay buffer (50 mM TRIS, pH 7.5, 10 mM NaCl, 250 mM sucrose, 5 mM MgCl₂, 1 mM EDTA, 1 mM DTT, and 2 mM ATP) and lysed by sonication. Lysates were centrifuged at 17,000 g for 10 min at 4 °C. Protein concentration was determined using the BCA protein assay (Thermo Fisher Scientific). 25 μg of total protein of cell lysates was transferred to a 96-well black plate (Corning Costar) and then the fluorogenic Suc-LLVY-AMC (100 μM, Enzo) substrate was added to lysates. Fluorescence intensity (340 nm excitation, 440 nm emission) was monitored using an Infinite F Plex (Tecan) every 20 min for 1.5 h at 37 °C and the data were analyzed by GraphPad Prism. Gating strategy is shown in the Supplementary information file.

We analyzed the proteasome activities in 25–50 μg protein extracts. The assay buffer consists of 50 mM Tris (pH 7.5), 2.5 mM EGTA, 20% glycerol, 1 mM DTT, 0.05% NP-40, and 25 μM substrate. Substrates Ac-nLPnLD-AMC, Bz-VGR-AMC and Suc-LLVY-AMC were from Enzo life science [43 (link), 68 (link)]. MG132 was used at a final concentration of 10 μM to block proteasome activities as negative controls. Fluorescence was read at 5 min intervals for 2 h, at an excitation wavelength of 380 nm and an emission wavelength of 460 nM, n ≥ 3 per group. Data were activities normalized to total protein and control in the assay.