MDA-MB-231, HCC1143, and HCC1937 breast cancer cells were purchased from the Korean Cell Line Bank (Seoul, Korea). Hep3B, Huh7, LCSC, HepG2, and PLC/PRF/5 hepatic cancer cells were a kind gift of Dr. Roberto Gedaly (College of Medicine, University of Kentucky). All other established cell lines were obtained from the American Type Culture Collection (ATCC, Rockville, MD). All cells were cultured according to the manufacturer’s protocol in 5% CO2 in medium. Cultured cell lines were tested for Mycoplasma contamination routinely every 6 months. Specifically, H23 and H727 cells were tested twice in the course of performing the experiments described within this publication (Supplementary Fig. S4 ). Inhibitors of UPS pathways used in this study were purchased from commercial vendors: carfilzomib (LC Laboratories, Woburn, MA), bortezomib (ChemieTek, Indianapolis, IN), MG-132 (EMD Millipore, San Diego, CA), PYR-41 (ApexBio, Houston, TX), and P5091 (ApexBio, Houston, TX). The following proteasome fluorogenic substrates were used: Suc-LLVY-AMC (Bachem, Torrance, CA; I-1395), Ac-WLA-AMC (Boston Biochem, Cambridge, MA; S-330), Ac-nLPnLD-AMC (Bachem; I-1850), Ac-RLR-AMC (Boston Biochem; S-290), Ac-ANW-AMC (Boston Biochem; S-320), and Ac-PAL-AMC (Boston Biochem; S-310). Human recombinant Interferon-γ was purchased from eBioscience (San Diego, CA).
Ac nlpnld amc
Manufactured by Bachem
Ac-nLPnLD-AMC is a peptide substrate that can be used in fluorescence-based enzyme activity assays. It contains the sequence Ac-norleucine-leucine-proline-norleucine-leucine-aspartic acid-7-amino-4-methylcoumarin, which is cleaved by certain proteases, resulting in the release of the fluorescent 7-amino-4-methylcoumarin moiety.
Automatically generated - may contain errors
Lab products found in correlation
Suc llvy amc, by Bachem (2 mentions)
Mg132, by Merck Group (1 mentions)
Bortezomib, by Chemietek (1 mentions)
Mda mb 231, by Korean Cell Line Bank (1 mentions)
Hcc1143, by Korean Cell Line Bank (1 mentions)
Hcc1937, by Korean Cell Line Bank (1 mentions)
Pyr 41, by Apexbio (1 mentions)
Ac rlr amc, by R&D Systems (1 mentions)
Ac anw amc, by R&D Systems (1 mentions)
Ac pal amc, by R&D Systems (1 mentions)
Carfilzomib, by LC Laboratories (1 mentions)
Chloroquine, by Merck Group (1 mentions)
Verteporfin, by Merck Group (1 mentions)
Everolimus, by InvivoGen (1 mentions)
Marizomib, by Merck Group (1 mentions)
Bafilomycin a1, by InvivoGen (1 mentions)
Boc lstr amc, by Bachem (1 mentions)
3 protocols using ac nlpnld amc
1
Comprehensive Cell Line Characterization
2
Proteasome Activity Assay
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Proteasome
activity was
determined using Ac-nLPnLD-AMC (Bachem) for caspase-like (β1)
activity, Ac-WLR-AMC (custom, Anaspec, Freemont, CA) for trypsin-like
(β2) activity, and Ac-WLA-AMC (custom, Anaspec, Freemont, CA)
for chymotrypsin-like (β5) activity. Compounds with 3-fold dilutions
from 10 μM (10 concentration points in duplicate) were plated
out into black 1536-well plates. The reaction was initiated by adding
the AMC substrates, PA28αβ, and 20S proteasome sequentially
to the plates. 1 nM Pf20S proteasome, 24 nM PA28αβ,
and 15 μM AMC substrates were incubated in the assay buffer
(50 mM Tris, 5 mM MgCl2, 1 mM DTT, 0.01% BSA, pH 7.4) at
37 °C for 1 or 2 h. Release of AMC fluorophore (excitation, 360
nm; emission, 450 nm) was measured using a fluorescence microplate
reader (FLUOstar, BMG Labtech). Slopes of AMC formation over the measurement
period were determined for each compound concentration to assess the
inhibitory effect (i.e., end-point assay). The same set of compounds
was also tested for inhibitory activity against human 20S constitutive
proteasome and immunoproteasome (Boston Biochem).
activity was
determined using Ac-nLPnLD-AMC (Bachem) for caspase-like (β1)
activity, Ac-WLR-AMC (custom, Anaspec, Freemont, CA) for trypsin-like
(β2) activity, and Ac-WLA-AMC (custom, Anaspec, Freemont, CA)
for chymotrypsin-like (β5) activity. Compounds with 3-fold dilutions
from 10 μM (10 concentration points in duplicate) were plated
out into black 1536-well plates. The reaction was initiated by adding
the AMC substrates, PA28αβ, and 20S proteasome sequentially
to the plates. 1 nM Pf20S proteasome, 24 nM PA28αβ,
and 15 μM AMC substrates were incubated in the assay buffer
(50 mM Tris, 5 mM MgCl2, 1 mM DTT, 0.01% BSA, pH 7.4) at
37 °C for 1 or 2 h. Release of AMC fluorophore (excitation, 360
nm; emission, 450 nm) was measured using a fluorescence microplate
reader (FLUOstar, BMG Labtech). Slopes of AMC formation over the measurement
period were determined for each compound concentration to assess the
inhibitory effect (i.e., end-point assay). The same set of compounds
was also tested for inhibitory activity against human 20S constitutive
proteasome and immunoproteasome (Boston Biochem).
3
Proteasome and Autophagy Inhibitors
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Ac-nLPnLD-amc, Boc-LSTR-amc and Suc-LLVY-amc peptides were purchased from Bachem AG (Bubendorf, CHE). Marizomib, chloroquine and verteporfin were from Sigma (St. Louis, MO), bortezomib was from Tebu bio (Le Perray en Yvelines, France), bafilomycin A1 and everolimus were from Invivogen (San Diego, CA, USA).
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