The proteasome activity was measured using a Proteasome-Glo Chymotrypsin-like Cell-Based Assay kit (Promega). Neuronally differentiated PC12 cells were seeded in 96-well culture plates at a density of 2.5×10 4 cells per well. After differentiation, the medium was replaced, and the neuronally differentiated PC12 cells were treated with ACA or its analogs for 1 to 24 h. After incubation, cells were assayed according to the manufactures protocol.
Proteasome glo chymotrypsin like cell based assay kit
Manufactured by Promega
Sourced in United States
The Proteasome-Glo Chymotrypsin-like Cell-Based Assay kit is a luminescent-based assay used to measure the chymotrypsin-like activity of the proteasome in living cells. The kit provides a reagent that generates a luminescent signal proportional to the amount of chymotrypsin-like proteasome activity present in the sample.
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Lab products found in correlation
Bilirubin, by Merck Group (1 mentions)
Biliverdin, by Merck Group (1 mentions)
Ha ubvs, by R&D Systems (1 mentions)
Anti ubiquitin p4d1, by Santa Cruz Biotechnology (1 mentions)
Z leu leu glu amc, by R&D Systems (1 mentions)
Enhanced chemiluminescence ecl reagent, by Santa Cruz Biotechnology (1 mentions)
Suc leu leu 5 al tyr aminomethylcoumarin suc llvy amc, by R&D Systems (1 mentions)
Anti ha tag, by Bioworld Technology (1 mentions)
K48 linked tetraubiquitin, by R&D Systems (1 mentions)
Mts assay kit celltiter 96 aqueous one solution reagent, by Promega (1 mentions)
Anti gapdh, by Bioworld Technology (1 mentions)
Synergy ht microplate reader, by Agilent Technologies (1 mentions)
Human 19s proteasome, by R&D Systems (1 mentions)
Matrigel, by BD (1 mentions)
Transwell chamber, by Corning (1 mentions)
Annexin 5 fitc apoptosis detection kit, by Keygen Biotech (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Beyotime (1 mentions)
6 protocols using proteasome glo chymotrypsin like cell based assay kit
1
Proteasome Activity in Differentiated PC12 Cells
2
Proteasome Inhibition and Ubiquitination Assays
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Bilirubin and biliverdin were purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA). Other agents used include NEM (Sigma-Aldrich Inc.), the Proteasome-Glo Chymotrypsin-like Cell-Based Assay kit (Promega Bioscience, Madison, WI, USA), Suc-Leu-Leu-Val-Tyr-aminomethylcoumarin (Suc-LLVY-AMC), Boc-Leu-Arg-Arg- aminomethylcoumarin (Boc-LRR-AMC), Z-Leu-Leu-Glu-AMC, 20S and 26S human proteasome preparations, HA-Ub-VS, K48-linked tetraubiquitin, Ubiquitin-AMC (U550) (Boston Biochem, Cambridge, MA, USA), JC-1 (Beyotime, Shanghai, China) and MTS assay kit (CellTiter 96 Aqueous One Solution reagent) (Promega Corporation, Madison, WI, USA). Antibodies used in this study and their sources are: anti-ubiquitin (P4D1) (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), anti-K48-linkage specific polyubiquitin (D9D5), anti-Bax (D3R2M) (Cell Signaling Technology, Beverly, MA, USA), anti-GAPDH, anti-HA-tag (Bioworld Technology, Inc., Louis Park, MN, USA) and anti-Tau-1, (Invitrogen, Carlsbad, CA, USA). Enhanced chemiluminescence (ECL) reagents were purchased from Santa Cruz Biotechnology Inc.
3
Proteasome Activity Measurement in SKOV3Trip2 Cells
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SKOV3Trip2 cells were plated in white-walled 96-well plates (2,000 cells/well), allowed to attach overnight, and exposed to DMSO, LDE225, GSI-I, Compound E or bortezomib, in quadruplicate, at the indicated doses for 24 hours. Proteasome activity was measured in these treated cells using the Proteasome-Glo Chymotrypsin-Like Cell-Based Assay kit (Promega, Madison, WI). One hundred μl of Proteasome-Glo reagent was added to the 100 μl of media present in each well, mixed for 2 minutes at 700 rpm using a plate shaker and incubated at room temperature for 2 hours. Luminescence was measured in each well using a Synergy HT Microplate Reader (Biotek).
4
Chymotrypsin Activity in Melanoma Cells
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Chymotryspin activity in melanoma cells was measured using the Proteasome-Glo™ Chymotrypsin-like cell based assay kit (Promega) according to the manufacturer’s instructions.
5
Proteasome Inhibition Assay with Bortezomib
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The Proteasome-Glo™ Chymotrypsin-like Cell-Based Assay Kit (Promega Corporation, Madison, WI, USA) was used to measure the proteasome inhibition activity of bortezomib. This assay used a luminogenic proteasome substrate containing succinyl-leucine-leucine-valine-tyrosine-aminoleuciferin in a buffer optimized for cell permeabilization, proteasome and luciferase activity.17 (link),18 (link) Adding a single Proteasome-Glo™ Cell-Based Reagent in an "add-mix-measure" format resulted in proteasomal cleavage of the substrate and generation of a luminescent signal produced by the luciferase reaction. Following the cleavage by the proteasome, the substrate for luciferase (aminoluciferin) was released, allowing the luciferase reaction to proceed and produce light. Ten minutes after adding various substrates, the luminescence was measured using a luminometer and expressed in relative luminescent units.17 (link),18 (link)
6
RTHF Inhibits Cancer Cell Proliferation
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Radix Tetrastigma Hemsleyani Flavone (RTHF) was synthesized in the Key Laboratory Research and Development of New Drugs of Traditional Chinese Medicine of Zhejiang Province. Our experiments used the following: FBS and RPMI 1640 (HyClone), MTT (Beyotime), Annexin V-FITC Apoptosis Detection Kit (Keygen), Transwell chamber (Corning, USA), Matrigel (BD Biosciences USA), Proteasome-Glo Chymotrypsin-like Cell-Based Assay kit, DUB-Glo Protease Assay kit (Promega, USA), human 19S proteasome (Boston Biochem), and the antibodies to Bax, Bcl-2, Caspase-3/9, ub-prs, USP14, UCHL5, and POH1 (CST).
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