Muscles were homogenized in NP40 cell lysis buffer (Invitrogen #FNN0021) and protein quantitation determined by using the Bio-Rad protein assay. Subsequently, muscle extracts were then all normalized to the same final protein concentration (1 mg/mL) by dilution with additional NP40 cell lysis buffer. 10 μL of the normalized NP40 muscle extracts were then used for the measurement of proteasome activity with the Proteasome-Glo 3-substrate cell-based assay system (Promega #G1180), as previously done (Hunt et al., 2021 (link); Rai et al., 2021a (link)). Specifically, 100 μL of the three different substrate reagents (to measure caspase-like, trypsin-like, and chymotrypsin-like proteolytic activities) were added individually to plates with 10 μL of the normalized NP40 muscle extracts. The plates were then incubated for 30 min at room temperature and the luminescence read on a Tecan Infinite 200 PRO plate reader. To ensure that the observed proteolytic activities indeed depend on the proteasome, some samples were treated with MG132 at a final concentration of 50 μM: this was added to the muscle extracts just before addition of the Proteasome-Glo3 substrates.
Proteasome glo 3 substrate cell based assay system
Manufactured by Promega
Sourced in United States
The Proteasome-Glo 3-substrate cell-based assay system is a laboratory equipment product designed to measure proteasome activity in cells. It utilizes a luminescent-based approach to quantify the chymotrypsin-like, trypsin-like, and caspase-like proteasome activities simultaneously.
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5 protocols using proteasome glo 3 substrate cell based assay system
1
Proteasome Activity in Muscle Extracts
2
Proteasome Activity Quantification in HGPS
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The proteasome activities were determined in HGPS and control cells by using the Proteasome‐Glo™ 3‐Substrate Cell‐Based Assay System (Promega) that allows to measure the chymotrypsin‐like, trypsin‐like, or caspase‐like protease activity associated with the proteasome complex in cultured cells. The Proteasome‐Glo™ Cell‐Based Reagents each contain a specific luminogenic proteasome substrate. These peptide substrates are Suc‐LLVY aminoluciferin (succinyl‐leucine‐leucine‐valine‐tyrosine‐aminoluciferin), Z‐LRR‐ aminoluciferin (Z‐leucine‐arginine‐arginine‐aminoluciferin) and Z‐nLPnLD‐aminoluciferin (Z‐norleucine‐proline‐norleucine‐aspartate‐aminoluciferin) for the chymotrypsin‐like, trypsin‐like and caspase‐like activities, respectively. Cells (10,000 cells/well) were cultured in 100 μl/well in a 96‐well plate at 37°C, 5% CO2. The plate was allowed to equilibrate to 22°C before 100 μl/well of substrate and luciferin detection reagent was added. The contents of the wells were mixed at 700 rpm using a plate shaker for 2 min and incubated at room temperature for 10 min. Following cleavage by the proteasome, the substrate for luciferase (aminoluciferin) is released, allowing the luciferase reaction to proceed and produce light. Luminescence is determined as relative light units (RLUs) using a GloMax‐Multi Detection System: Luminometer (Promega, USA).
3
Measuring Proteasome Activity in Cells
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SF were transfected and treated as described above. Proteasome activities were measured using the Proteasome-Glo 3-Substrate Cell Based Assay System (Promega) following the manufacturer`s instructions.
4
Proteasome Activity in Muscle Extracts
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Muscles were homogenized in NP40 cell lysis buffer (Invitrogen #FNN0021) and protein quantitation determined by using the Bio-Rad protein assay. Subsequently, muscle extracts were then all normalized to the same final protein concentration (1 mg/mL) by dilution with additional NP40 cell lysis buffer. 10 μL of the normalized NP40 muscle extracts were then used for the measurement of proteasome activity with the Proteasome-Glo 3-substrate cell-based assay system (Promega #G1180), as previously done (Hunt et al., 2021 (link); Rai et al., 2021a (link)). Specifically, 100 μL of the three different substrate reagents (to measure caspase-like, trypsin-like, and chymotrypsin-like proteolytic activities) were added individually to plates with 10 μL of the normalized NP40 muscle extracts. The plates were then incubated for 30 min at room temperature and the luminescence read on a Tecan Infinite 200 PRO plate reader. To ensure that the observed proteolytic activities indeed depend on the proteasome, some samples were treated with MG132 at a final concentration of 50 μM: this was added to the muscle extracts just before addition of the Proteasome-Glo3 substrates.
5
Proteasome Activity Profiling in α-Synuclein Cells
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The enzymatic activities of the chymotrypsin-, trypsin- and caspase-like proteasome were measured using the Proteasome-Glo™ 3-Substrate Cell-Based Assay System (Promega, USA) following the manufacturer’s instructions. Briefly, inducible PC12/A53T-α-syn cells were treated with or without 1 μg/ml Dox for 24 h, followed by treatment with 100 μΜ Oxyphylla A, 0.1 μΜ MG132 for another 24 h. The cells were harvested and lysed with Proteasome-Glo™ Cell-Based Reagent. Cell lysates were assayed in triplicate for each proteasome. Suc-LLVY aminoluciferin (succinyl-leucine-leucine-valine-tyrosine-aminoluciferin) substrate was used for the measurement of chymotryptic-like activity, while Z-LRR-aminoluciferin (Z-leucine-argininearginine-aminoluciferin) substrate was applied for trypsin-like activity detection. Z-nLPnLD-aminoluciferin (Z-norleucine-proline-norleucine-aspartate-aminoluciferin) substrate was then employed for the analysis of caspase-like activity. After reagents were added and mixed by plate shaking, luminescence was recorded using the SpectraMax M5.
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