LPS (Escherichia coli, O127:B8) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Mas receptor antagonist A779 was obtained from AbBiotech (San Diego, CA, USA). ACE2 inhibitor MLN-4760 was a product from EMD Millipore (Darmstadt, Germany). SB203580 (a specific inhibitor of p38 MAPK), PD98059 (a specific inhibitor of ERK1/2) and SP600125 (a specific inhibitor of JNK) were purchased from Santa Cruz Biotechnology (Delaware, CA, USA). Rabbit anti-ACE2, anti-p50, anti-p65, and mouse anti-phospho-p50, anti-phospho-p65 and anti-IκBα antibodies were procured from Santa Cruz Biotechnology. Rabbit anti-p38 MAPK, anti-phospho-p38 MAPK, anti-ERK1/2, anti-phospho-ERK1/2, anti-/JNK, anti-phospho-JNK, horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG, and horse anti-mouse IgG antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). TNF-α and IL-1β kits were purchased from Invitrogen (Eugene, OR, USA). AngII and Ang-(1-7) enzyme-linked immunosorbent assay (ELISA) kits were from Kamiya Biomedical (Seattle, WA, USA).
Mln 4760
Manufactured by Merck Group
Sourced in United States, Germany
MLN-4760 is a laboratory instrument designed for use in research and scientific applications. It is a multi-functional device that can perform various analytical tasks. The core function of MLN-4760 is to assist researchers in their investigations, but a detailed description of its intended use cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Z pro prolinal, by Merck Group (3 mentions)
Lisinopril, by Merck Group (2 mentions)
Aminopeptidase inhibitor, by Merck Group (2 mentions)
Pepstatin a, by Merck Group (2 mentions)
Leupeptin, by Merck Group (2 mentions)
Horse anti mouse igg, by Cell Signaling Technology (1 mentions)
Sb203580, by Santa Cruz Biotechnology (1 mentions)
Lps escherichia coli o127 b8, by Merck Group (1 mentions)
Pd98059, by Santa Cruz Biotechnology (1 mentions)
Anti phospho p65, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti p38 mapk, by Cell Signaling Technology (1 mentions)
Il 1β kit, by Thermo Fisher Scientific (1 mentions)
Sp600125, by Santa Cruz Biotechnology (1 mentions)
Anti iκbα, by Santa Cruz Biotechnology (1 mentions)
Anti phospho p38 mapk, by Cell Signaling Technology (1 mentions)
Anti p65, by Santa Cruz Biotechnology (1 mentions)
Anti phospho erk1 2, by Cell Signaling Technology (1 mentions)
Tnf α, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase hrp conjugated goat anti rabbit igg, by Cell Signaling Technology (1 mentions)
Anti erk1 2, by Cell Signaling Technology (1 mentions)
17 protocols using mln 4760
1
Investigating Inflammatory Signaling Pathways
2
Lipoxin A4 Receptor Modulation in LPS-Induced Inflammation
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LPS (Escherichia coli 055:B5) was from Sigma-Aldrich (St. Louis,
MO, USA). Lipoxin A4 (LXA4) (5S,
6R, 15S-trihydroxy-7E,
9E, 11Z,
13E-eicosa-tetraenoic acid) was from Cayman Chemical (Ann
Arbor, MI, USA), BOC-2 (LXA4 receptor antagonist) was from GenScript
(Piscataway, NJ, USA), A779 (selective inhibitor of the Mas receptor) was from
Abcam (Cambridge, MA, USA), and MLN-4760 (ACE2 antagonist) was from Merck
Millipore (Cambridge, UK). The myeloperoxidase (MPO) kit was from Nanjing
Jiancheng Bioengineering Institute (Nanjing, China). The ACE2 activity assay kit
was from Genmed Scientifics (Arlington, VA, USA). ELISA kits for ACE2,
Ang-(1-7), inflammatory factors (TNF-α, IL-1β, and IL-10), and ROS were from
Cloud-Clone (Houston, TX, USA). Nuclear Transfer Assay Kit of NF-κB Activation
was from Beyotime Biotechnology (Shanghai, China). The primary Abs against
Ang-(1-7), Mas, and β-actin were from Cloud-Clone. Primary Abs against NF-κB
p65, p-p65, and inhibitor of NF-κB (IκB-α) were from Cell Signaling Technology
(Danvers, MA, USA). The Eastep Super Total RNA Extraction Kit, Easy Script
First-Strand cDNA Synthesis Super Mix Kit, and TransStarat Tip Green qPCR Super
Mix Kit were all from Promega (Madison, WI, USA).
MO, USA). Lipoxin A4 (LXA4) (5S,
6R, 15S-trihydroxy-7E,
9E, 11Z,
13E-eicosa-tetraenoic acid) was from Cayman Chemical (Ann
Arbor, MI, USA), BOC-2 (LXA4 receptor antagonist) was from GenScript
(Piscataway, NJ, USA), A779 (selective inhibitor of the Mas receptor) was from
Abcam (Cambridge, MA, USA), and MLN-4760 (ACE2 antagonist) was from Merck
Millipore (Cambridge, UK). The myeloperoxidase (MPO) kit was from Nanjing
Jiancheng Bioengineering Institute (Nanjing, China). The ACE2 activity assay kit
was from Genmed Scientifics (Arlington, VA, USA). ELISA kits for ACE2,
Ang-(1-7), inflammatory factors (TNF-α, IL-1β, and IL-10), and ROS were from
Cloud-Clone (Houston, TX, USA). Nuclear Transfer Assay Kit of NF-κB Activation
was from Beyotime Biotechnology (Shanghai, China). The primary Abs against
Ang-(1-7), Mas, and β-actin were from Cloud-Clone. Primary Abs against NF-κB
p65, p-p65, and inhibitor of NF-κB (IκB-α) were from Cell Signaling Technology
(Danvers, MA, USA). The Eastep Super Total RNA Extraction Kit, Easy Script
First-Strand cDNA Synthesis Super Mix Kit, and TransStarat Tip Green qPCR Super
Mix Kit were all from Promega (Madison, WI, USA).
3
Assay for Renin Activity in Tissue
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Metabolic RAS analysis was performed by Attoquant Diagnostics GmbH, Vienna, Austria. Renal biopsies of mice and humans were extracted and homogenized in phosphate-buffered saline (PBS) using low-energy sonication. The metabolism of spiked Ang I, Ang II (Fisher Scientific) or recombinant murine AGT (Sino Biological Inc.) for the renin activity assay, respectively, was assayed in homogenates after ex vivo incubation at 37 °C in presence or absence of specific enzyme inhibitors. Lisinopril (10 μM, Sigma-Aldrich), chymostatin (10 μM, Sigma-Aldrich), Z-Pro-Prolinal (20 μM, Sigma-Aldrich), MLN-4760 (10 μM, Merck-Millipore), DL-thiorphan (100 μM, Sigma-Aldrich). Aminopeptidase Inhibitor (10 μM, Sigma-Aldrich) was added to all samples.
The enzyme activity was calculated by determining the inhibitor sensitive part of product formation in incubated samples and is presented as a product formation in ng of the corresponding angiotensin product per μg protein per hour. In order to proof linearity of the assay in dependence of enzyme abundance, Ang I and Ang II metabolism were assayed in serial diluted kidney homogenates (Supplementary Fig. 6 ). All experiments were performed in the linear range of the assay.
The enzyme activity was calculated by determining the inhibitor sensitive part of product formation in incubated samples and is presented as a product formation in ng of the corresponding angiotensin product per μg protein per hour. In order to proof linearity of the assay in dependence of enzyme abundance, Ang I and Ang II metabolism were assayed in serial diluted kidney homogenates (
4
Measuring Ang-(1-7) Formation in Tissues
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To prove the feasibility of measuring Ang-(1–7) formation in APA and APA-adjacent tissues, we measured Ang-(1–7) formation in a metabolic assay. Briefly, tissue protein extracts were spiked with Ang II, in the presence and absence of MLN-4760 (Merck-Millipore, Vienna, Austria), an established ACE-2 inhibitor. Positive control samples included recombinant human (rh)ACE-2 (1 ng rhACE-2/μg protein) and a sample of proteins from tissues spiked with rhACE-2; both of which were analysed in the presence and absence of MLN-4760. Protein concentration was measured with the Bradford method and was set equal for each sample.
5
Quantifying ACE2 Enzymatic Activity
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ACE2 activity was measured using the ACE2 specific FRET substrate (Mca-APK(Dnp) (Enzo Life Sciences, Exeter, UK) as previously described [10] . Recombinant ACE2 (440 -6 ng/ml) (R&D systems, Cambridge, UK) or CSF (50 μl undiluted) was incubated with the FRET substrate (10 μM) diluted in assay buffer (75 mM Tris, 1M NaCl, pH7.5) for 3 h at 37 C. Recombinant ACE2 and CSF samples were assayed in duplicate in the presence or absence of an ACE2 specific inhibitor (MLN4760) (10 μM) (Merck, Nottingham, UK) diluted in distilled water for 10 mins at 37°C prior to the addition of the fluorogenic substrate. ACE2 activity was determined by subtracting the fluorescence in the inhibited from the untreated wells.
Cleavage of the ACE2 FRET peptide was measured using a microplate reader (BMG Labtech, Aylesbury, UK) at excitation/emission wavelength 330/390nm. A serial dilution of recombinant human ACE2 and measurements on carry-over samples across all plates was used to minimise variation between plates. ACE2 activity was expressed in relative fluorescence units (r.f.u).
Cleavage of the ACE2 FRET peptide was measured using a microplate reader (BMG Labtech, Aylesbury, UK) at excitation/emission wavelength 330/390nm. A serial dilution of recombinant human ACE2 and measurements on carry-over samples across all plates was used to minimise variation between plates. ACE2 activity was expressed in relative fluorescence units (r.f.u).
6
Investigating ACE-2 Role in Stroke Protection
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To investigate contributions of ACE‐2 in hPMSC‐mediated stroke protection, hPMSCs were treated with 10 μM MLN‐4760 (Sigma Aldrich), to selectively inhibit ACE‐2 activity.24 MLN‐treated hPMSC were PBS washed after 48 hours treatment and injected (5 × 105 cells in 500 μL HBSS) into the MCAO mice.
7
Measuring Anti-hACE2 mAbs Catalytic Effects
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To measure the effect of anti-hACE2 mAbs on the catalytic activity of hACE2, various concentrations of 2G7A1, 05B04, 05H02 and 05B04LC/05D06HC (2 μg ml−1, 10 μg ml−1 and 50 μg ml−1) were mixed with hACE2 at 0.2 μg ml−1. Human ACE2 enzymatic activity was then measured using the ACE2 Inhibitor Screening Assay Kit (BPS Bioscience) following the manufacturer’s instructions. The intensity of the fluorescent product of the hACE2 reaction product was detected at 555 nm/585 nm (excitation/emission) with Clariostar Plus Microplate Reader (BMG Labtech). MLN-4760 (Sigma, #5306160001) served as a positive control ACE2 inhibitor.
8
Protease Activity of ACE2 with Peptide Substrates
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To compare the relative proteolytic activity of ACE2 with different substrates, various concentrations of nonfluorogenic BK, desBK or Ang II peptides were added to the reaction mixture with fluorogenic peptides as competitive substrates. As mentioned above, 50 µl of ACE2 at 0.4 µg/ml was preincubated with buffers or SARS-CoV-2 RBD protein at a final concentration of 125 nm for 20 min. Subsequently, 50 µl of fluorogenic peptides at 20 μm supplemented with a serial dilution of nonfluorogenic BK, desBK or Ang II peptides ranging 640 μm to 80 μm were added to initiate enzymatic cleavage. In addition, 10 μm of ACE2 specific inhibitor MLN-4760 (Sigma-Aldrich) was used as positive control to block ACE2 activity in the absence or presence of SARS-CoV-2 spike(15 nm ), SARS-CoV-2 RBD(125 nm ) or SARS-CoV RBD (125 nm ) proteins. Various protease inhibitors at a final concentration of 40 μm such as AEBSF, pepstatin A and leupeptin (Sigma-Aldrich) were also used to verify the specific activity of ACE2 enzyme. Fluorescence changes were measured at 1 min intervals for 1 h at 26 degree as the initial velocity conditions. The dose dependent inhibition was fitted with a competitive inhibition model to obtain IC50.
9
Inhibiting ACE-2 in hPMSCs for Stroke Protection
10
Inflammasome Activation in MDMi Cells
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For inflammasome activation experiments, MDMi were primed with 200 ng/ml of ultrapure LPS (E.Coli 0111:B4, Invivogen) for 3 h or 50 μg of S-clamp or F-clamp for 6 h. Cells were washed in after priming to remove residual LPS or S-Clamp and cells were stimulated with conventional NLRP3 inflammasome activators ATP (5 mM, Sigma) and nigericin (10 μM, Invivogen), or fibrillar α-synuclein (10 μM, Proteos), S-Clamp (2–50 μg) or SARS-CoV-2 isolates (MOI 0.1, 1) for the indicated time. For priming studies, MDMis were pre-treated with the NF-kB inhibitor, Bay 11-7082 (3 μM, Sigma), before stimulation with S-clamp and the addition of ATP, nigericin or α-synuclein. For inhibition studies, MCC950 (10 μM), VX-765 (20 μM, Invivogen) and MLN-4760 (1,10 μM, Sigma) were added after the priming step. At the end of treatment, the supernatant was collected and stored at −80 °C until analysis by enzyme-linked immunosorbent assay (ELISA) or western blotting.
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