Ac rlr amc

Manufactured by R&D Systems

Ac-RLR-AMC is a fluorogenic substrate for the detection and measurement of caspase-3 activity. It contains the amino acid sequence Ac-Arg-Leu-Arg labeled with the fluorophore 7-amino-4-methylcoumarin (AMC). The cleavage of the substrate by caspase-3 results in the release of the fluorescent AMC moiety, which can be detected using a fluorometer.

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Lab products found in correlation

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 nlpnld amc, by Bachem (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) Mg132, by Merck Group (1 mentions) Suc llvy amc, by Bachem (1 mentions) Spectramax m5e microplate reader, by Molecular Devices (1 mentions) Ac gpld amc, by Enzo Life Sciences (1 mentions) Black walled 96 well plate, by Greiner (1 mentions) Z leu leu glu amc, by R&D Systems (1 mentions) Suc llvy amc, by R&D Systems (1 mentions) Spectramax m5, by Molecular Devices (1 mentions) Epoxomicin, by R&D Systems (1 mentions) Protein assay, by Bio-Rad (1 mentions) Z lle amc, by R&D Systems (1 mentions)

4 protocols using ac rlr amc

Comprehensive Cell Line Characterization

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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).

Lee M.J., Miller Z., Park J.E., Bhattarai D., Lee W, & Kim K.B. (2019). H727 cells are inherently resistant to the proteasome inhibitor carfilzomib, yet require proteasome activity for cell survival and growth. Scientific Reports, 9, 4089.

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Proteasome Activity Assay Protocol

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Proteasome activity was assessed as previously described 32. 10 μg of tissue protein lysates was added to proteasome activity assay buffer (50 mM Tris–HCl, pH 7.5) along with 10 μM of either chymotrypsin (Suc‐LLVY‐AMC) (Calbiochem, cat. no. 539142), trypsin (Ac‐RLR‐AMC) (Boston Biochem, cat. no. s290) or caspase (Ac‐GPLD‐AMC) (Enzo Life Sciences, cat no. BML‐AW9560‐0005) fluorogenic proteasome substrates. Reactions were incubated at 37°C protected from light for 3 h. Following incubation, samples were transferred to black walled 96‐well plates (Greiner Bio‐One, cat no. 655097). Release of free 7‐amino‐4‐methylcoumarin (AMC) was determined using a SpectraMax M5e Microplate Reader (Molecular Devices) with excitation at 380 nm and emission recorded at 460 nm. Reported values are the fold increase over a no‐protein control. For treatment with bortezomib, 10 μg of protein lysate was preincubated with either 2 mM bortezomib or an equivalent volume of DMSO at 4°C for 1 h before adding reaction buffer containing substrate.

Jenkins E.C., Shah N., Gomez M., Casalena G., Zhao D., Kenny T.C., Guariglia S.R., Manfredi G, & Germain D. (2020). Proteasome mapping reveals sexual dimorphism in tissue‐specific sensitivity to protein aggregations. EMBO Reports, 21(4), e48978.

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Proteasome Activity Assay in Cardiac Cells

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Snap-frozen tissues and cultured NRCMs were homogenized on ice in cytosolic extraction buffer (50 mM Tris-HCl pH 7.5, 250 mM Sucrose, 5 mM MgCl₂, 0.5 mM EDTA, and 1 mM DTT). Protein concentrations were determined with bicinchoninic acid (BCA) reagents (Pierce) and equally concentrated in proteasome assay buffer (50 mM Tris-HCl pH 7.5, 40 mM KCl, 5 mM MgCl₂, and 1 mM DTT). Chymotrypsin-like, trypsin-like, and caspase-like activities were determined in the presence of 28 μM (chymotrypsin) or 14 μM (trypsin and caspase) ATP, utilizing the following fluorogenic substrates: Suc-LLVY-AMC (18 μM, Boston Biochem #S280), Ac-RLR-AMC (45 μM, Boston Biochem #S290), and Z-Leu-Leu-Glu-AMC (40 μM, Boston Biochem #S230), respectively. The plate was read at an excitation wavelength of 380 nm and an emission wavelength of 460 nm using a Spectramax M5 (Molecular Devices). Activity was combined, using a 50:25:25 ratio for relative contribution of chymotrypsin, trypsin, and caspase-like activities⁴⁹.

Ranek M.J., Oeing C., Sanchez-Hodge R., Kokkonen-Simon K.M., Dillard D., Aslam M.I., Rainer P.P., Mishra S., Dunkerly-Eyring B., Holewinski R.J., Virus C., Zhang H., Mannion M.M., Agrawal V., Hahn V., Lee D.I., Sasaki M., Van Eyk J.E., Willis M.S., Page R.C., Schisler J.C, & Kass D.A. (2020). CHIP phosphorylation by protein kinase G enhances protein quality control and attenuates cardiac ischemic injury. Nature Communications, 11, 5237.

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Measuring Proteasomal Activity in Tissue Slices

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Tissue slices (6 pooled slices/sample) were lysed in ice-chilled buffer containing 20mM Tris-HCl pH7.5, 1mM MgCl₂, 1mM EDTA and 1mM DTT. Lysates were cleared by centrifugation and protein concentration was measured with the Bio-Rad protein assay. Chymotryptic, tryptic and caspase-like proteasomal activities were determined in lysates by cleavage of amino-4-methylcoumarin (AMC) from fluorogenic peptide substrates Suc-LLVY-7-AMC, Ac-RLR-AMC and Z-LLE-AMC (all Boston Biochem, Cambridge, MA), respectively. 20 μg total protein was pre-incubated at 37°C for 2min before addition of 10μM substrate peptides. Thereafter, AMC fluorescence was continuously measured for lmin at 37°C by using an Ex/Em 380/460nm filter set on a F-2500 fluorescence spectrophotometer (Hitachi, Tokyo, Japan). AMC fluorescence generated over time was considered directly proportional to proteasomal catalytic activity in samples. Verifying functionality of the assay, addition of 10μM proteasome inhibitor lactacystin (Boston Biochem) to the reaction completely inhibited generation of AMC fluorescence. Changes in proteasomal activities in slices after ischemia, after induction of ROS production with H₂O₂ or X/XO, or after treatment with the proteasome inhibitor epoxomicin (Boston Biochem) were calculated relative to control conditions.

Kahles T., Poon C., Qian L., Palfini V., Srinivasan S.P., Swaminathan S., Blanco I., Rodney-Sandy R., Iadecola C., Zhou P, & Hochrainer K. (2020). Elevated post-ischemic ubiquitination results from suppression of deubiquitinase activity and not proteasome inhibition. Cellular and molecular life sciences : CMLS, 78(5), 2169-2183.

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