Journal: bioRxiv

Article Title: A Degenerate PCNA-Interacting Peptide (DPIP) box targets RNF168 to replicating DNA to limit 53BP1 signaling

doi: 10.1101/2021.03.17.435897

Figure Lengend Snippet: (A) Domain organization of RNF168 indicating key functional domains and a degenerate PIP-like sequence residing in a disordered C-terminal region. The protein disorder profile was generated using the Protein Disorder Prediction (PrDOS) tool at http://prdos.hgc.jp/cgi-bin/top.cgi . (B) Replicate plates of H1299 cells were co-infected with adenovirus vectors encoding HA-PCNA and RNF168 WT or RNF168 ΔDPIP. 24 h post infection, some cultures were treated with HU (2mM, 2 h), camptothecin (CPT, 100 nM, 2 h), ATM inhibitor KU55933 100 nM, 2 h), or the WEE1 inhibitor MK1775 (10 μM, 2h). Chromatin extracts from the treated and untreated (control) cells were normalized for protein content and immunoprecipitated with anti-HA antibodies. Anti-HA immunoprecipitates were resolved on SDS-PAGE, transferred to nitrocellulose, and analyzed by immunoblotting with the indicated antibodies. (C) Replicate plates of RNF8 -/- U2OS cells were transiently infected with adenovirus vectors encoding FLAG-RNF168 WT, FLAG-RNF168 ΔDPIP, or FLAG-RNF168 ΔMIU2, or with a control ‘empty’ adenoviral vector. 48 h post-infection chromatin extracts were prepared, normalized for protein content and immunoprecipitated with anti-FLAG antibodies. Anti-FLAG immunoprecipitates were resolved on SDS-PAGE, transferred to nitrocellulose, and analyzed by immunoblotting with the indicated antibodies. (D) Isothermal titration calorimetry (left panel) was conducted by titrating synthetic peptide (p21, GRKRR QTSMTDFY HSKRRLIFS-amide where underlined residues denote PIP box; syringe, 150 - 300 μM) into a solution of PCNA (cell; 30 μM) in TBS. Control experiments using peptide injected into buffer alone showed minimal heats with no evidence of titration. Analysis of the isotherm yielded Kd = 76.3 nM and stoichiometry = 0.93 (Microcal Origin software). Replicate plates of H1299 cells were infected with adenovirus vectors encoding FLAG-RNF168 WT and HA-PCNA (or an ‘empty’ adenovirus vector for control). 36 h post-infection, chromatin extracts were prepared, normalized for protein content and immunoprecipitated with anti-FLAG antibody in the presence of different concentrations of peptides corresponding to the p21 or Polη PIP boxes. Anti-FLAG immunoprecipitates were resolved on SDS-PAGE, transferred to nitrocellulose, and analyzed by immunoblotting with the indicated antibodies (right panel) . (E) Replicate plates of H1299 cells were co-infected with adenovirus vectors encoding HA-PCNA and FLAG-RNF168 WT or HA-PCNA and RNF168 ΔDPIP. Some cells received an ‘empty’ adenovirus vector instead of HA-PCNA (for control). 36 h post-infection, some plates were treated with 2 mM HU for 2 h. Chromatin extracts were prepared, normalized for protein content and immunoprecipitated with anti-HA antibody in the presence or absence of the p21 PIP box peptide (1 mM). Anti-HA immune complexes were resolved on SDS-PAGE, transferred to nitrocellulose, and analyzed by immunoblotting with the indicated antibodies. (F) Replicate plates of H1299 cells were infected with adenovirus vectors encoding wild-type PCNA (HA-PCNA WT), ubiquitylation-resistant PCNA (HA-PCNA K164R), or a PCNA-ubiquitin fusion (HA-PCNA-Ub) in combination with FLAG-RNF168 WT adenovirus. Some cells received an ‘empty’ adenovirus vector instead of HA-PCNA viruses(for control). 36 h post-infection, chromatin extracts were prepared, normalized for protein content and immunoprecipitated with anti-HA antibody. Anti-HA immune complexes were resolved on SDS-PAGE, transferred to nitrocellulose, and analyzed by immunoblotting with the indicated antibodies. (G) H1299 cells were transiently co-transfected with expression constructs encoding HA-PCNA and FLAG-RNF168 WT or FLAG-RNF168 ΔDPIP. 48 h post-transfection cells were fixed, then stained with anti-HA and anti-FLAG antibodies prior to analysis by immunofluorescence confocal microscopy. The photographs are of representative cells co-expressing HA-PCNA and WT or ΔDPIP forms of FLAG-RNF168. The bar chart shows enumeration of cells containing HA-PCNA-co-localizing FLAG-RNF168 foci. Each data point represents the mean of results from 3 separate experiments and the error-bars represent the standard deviation. (H) RNF168 -/- U2OS cells were infected with adenoviral vectors encoding FLAG-RNF168 WT or FLAG-RNF168 ΔDPIP. 24 h post-infection cells were fixed and subject to proximity ligation assays to compare proximities of WT and ΔDPIP RNF168 with endogenous PCNA. The photographs are of representative DAPI-stained nuclei from each experimental condition. The bar chart showing enumeration of cells containing PCNA / FLAG-RNF168 PLA foci. Each data point represents the mean of results from 3 separate experiments and the error-bars represent the standard deviation. Ordinary one-way ANOVA demonstrated statistically significant difference between RNF168 WT and PIP (p = 0.0049).

Article Snippet: Ubiquitylation assays were performed in 25 mL reactions in which the components were added in the following order: dd H2O, 1x Energy regeneration solution (#B-10 R&D systems), 75 mM ubiquitin (#U-100H R&D systems) 1.6 mM FLAG-PCNA substrate (expressed and purified in bacteria), 0.1 mM E1 Ubiquitin Activating Enzyme (#E-304 R&D systems), 0.2 mM E2 conjugase (UbcH5c, #E2-627 R&D systems, or RAD6 #E2-613 R&D Systems), 0.2 mM E3 ligase (recombinant bacterial RAD18-RAD6 complex or RNF168 both purified in-house).

Techniques: Functional Assay, Sequencing, Generated, Infection, Control, Immunoprecipitation, SDS Page, Western Blot, Plasmid Preparation, Isothermal Titration Calorimetry, Injection, Titration, Software, Ubiquitin Proteomics, Transfection, Expressing, Construct, Staining, Immunofluorescence, Confocal Microscopy, Standard Deviation, Ligation

Journal: bioRxiv

Article Title: A Degenerate PCNA-Interacting Peptide (DPIP) box targets RNF168 to replicating DNA to limit 53BP1 signaling

doi: 10.1101/2021.03.17.435897

Figure Lengend Snippet: (A) Replicate plates of RNF8 +/+ and RNF8 -/- U2OS cells were infected with adenovirus vectors encoding RNF168 WT, RNF168 ΔDPIP, RNF168 super-PIP, and RNF168 Δ121-157, or with an empty adenovirus vector as control. After 23 h, one of the empty vector control plates was irradiated with UVC (60 J/m 2 ). All plates of cells were collected 24 h post-infection and fractionated to give chromatin and soluble extracts. Cell extracts were normalized for protein content, resolved by SDS-PAGE and transferred to nitrocellulose membranes prior to immunoblotting with the indicated antibodies. (B) Replicate cultures of U2OS cells were infected with adenovirus vectors encoding RNF168 WT, RNF168 ΔDPIP, RNF168 super-PIP, and RNF168 Δ121-157, or with an empty adenovirus vector as control. After 23 h cells were labelled with EdU, extracted with nonionic detergent to remove unbound MCM, fixed, and stained with anti-MCM2 (a marker for the MCM2-7 complex), PI (total DNA), and for EdU incorporation (active DNA synthesis). Cell cycle phases are defined by DNA content (PI-A) and DNA synthesis (Edu-A) in the upper plots. Nuclei containing loaded MCM2 in G1 and S phase are represented by blue and orange dots respectively. G1/G2/M phase cells negative for chromatin-loaded MCM2 are shown in grey. (C) Replicate plates of RAD18 +/+ and RAD18 -/- H1299 cells were infected with adenovirus vectors encoding different RNF168 variants (RNF168 WT, RNF168 ΔDPIP, RNF168 super-PIP, and RNF168 Δ121-157) in combination with RAD18 adenovirus, or with an empty adenovirus vector as control. After 22 h, two cultures were irradiated with UVC (60 J/m 2 ). All plates of cells were collected 24 h post-infection and fractionated to give chromatin and soluble extracts. Cell extracts were normalized for protein content, resolved by SDS-PAGE and transferred to nitrocellulose membranes for immunoblotting with the indicated antibodies. (D) Replicate plates of RAD18 +/+ and RAD18 -/- H1299 cells were sequentially transfected with HLTF-directed siRNA or with non-targeting control siRNA (siCon), then with CMV-FLAG RNF168 WT (or with an empty vector for control). 48 h post-transfection, some cultures were conditionally irradiated with UVC (20 J/m 2 ). After 2 h chromatin fractions were prepared and analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. (E) Purified PCNA substrate was incubated in vitro with recombinant RNF168 and recombinant UBCH5 individually or in combination, with recombinant RAD18-RAD6 complex, or with a combination of RAD18-RAD6 complex and RNF168 in the presence of E1, ubiquitin and an ATP-regenerating system. Reactions were terminated after 15 min or 30 min and products were separated on SDS-PAGE, transferred to nitrocellulose and analyzed by immunoblotting with the indicated antibodies.

Article Snippet: Ubiquitylation assays were performed in 25 mL reactions in which the components were added in the following order: dd H2O, 1x Energy regeneration solution (#B-10 R&D systems), 75 mM ubiquitin (#U-100H R&D systems) 1.6 mM FLAG-PCNA substrate (expressed and purified in bacteria), 0.1 mM E1 Ubiquitin Activating Enzyme (#E-304 R&D systems), 0.2 mM E2 conjugase (UbcH5c, #E2-627 R&D systems, or RAD6 #E2-613 R&D Systems), 0.2 mM E3 ligase (recombinant bacterial RAD18-RAD6 complex or RNF168 both purified in-house).

Techniques: Infection, Plasmid Preparation, Control, Irradiation, SDS Page, Western Blot, Staining, Marker, DNA Synthesis, Transfection, Purification, Incubation, In Vitro, Recombinant, Ubiquitin Proteomics

Journal: bioRxiv

Article Title: A Degenerate PCNA-Interacting Peptide (DPIP) box targets RNF168 to replicating DNA to limit 53BP1 signaling

doi: 10.1101/2021.03.17.435897

Figure Lengend Snippet: RNF168 and RAD18 both ubiquitinate histone H2A in the vicinity of DSB to promote 53BP1 signaling and NHEJ (left panel) and also ubiquitinate PCNA to promote TLS (right panel) . RAD18 additionally acts as a molecular chaperone for the RAD51D recombinase and promotes HR independently of its ubiquitin ligase activity (middle) . See ‘Discussion’ for details.

Article Snippet: Ubiquitylation assays were performed in 25 mL reactions in which the components were added in the following order: dd H2O, 1x Energy regeneration solution (#B-10 R&D systems), 75 mM ubiquitin (#U-100H R&D systems) 1.6 mM FLAG-PCNA substrate (expressed and purified in bacteria), 0.1 mM E1 Ubiquitin Activating Enzyme (#E-304 R&D systems), 0.2 mM E2 conjugase (UbcH5c, #E2-627 R&D systems, or RAD6 #E2-613 R&D Systems), 0.2 mM E3 ligase (recombinant bacterial RAD18-RAD6 complex or RNF168 both purified in-house).

Techniques: Ubiquitin Proteomics, Activity Assay

Journal: iScience

Article Title: Evolutionary conserved regulation of TFEB stability by the E3 ubiquitin ligase WWP2 modulates response to stress in vivo

doi: 10.1016/j.isci.2025.111838

Figure Lengend Snippet:

Article Snippet: recombinant human 6His-Ube1 , RD System , Cat# E-305.

Techniques: Virus, Recombinant, Ubiquitin Proteomics, Luciferase, Mutagenesis, Reverse Transcription, SYBR Green Assay, Plasmid Preparation, Software, Lysis

Journal: International Journal of Molecular Sciences

Article Title: A Natural Bioactive Peptide from Pinctada fucata Pearls Can Be Used as a Potential Inhibitor of the Interaction between SARS-CoV-2 and ACE2 against COVID-19

doi: 10.3390/ijms25147902

Figure Lengend Snippet: ( A ) Molecular docking of SARS-CoV-2 S spike protein and ACE2 protein. ( B ) Molecular docking of KKCH peptide and ACE2 protein. ( C ) The common binding pocket of KKCH peptides and the SARS-CoV-2 S spike protein. ( D ) The interaction sites between the SARS-CoV-2 S spike protein and ACE2 protein. ( E ) The interaction sites between KKCH peptides and ACE2 protein. The blue solid line represents hydrogen bonding, the gray dashed line represents hydrophobic interactions, and the yellow dashed line represents salt bridging.

Article Snippet: The ACE2 protein (no. CSB-MP866317HU) was a recombinant human protein purchased from CUSABIO (Wuhan, China).

Techniques: Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: A Natural Bioactive Peptide from Pinctada fucata Pearls Can Be Used as a Potential Inhibitor of the Interaction between SARS-CoV-2 and ACE2 against COVID-19

doi: 10.3390/ijms25147902

Figure Lengend Snippet: ( A ) Binding kinetics of KKCH with ACE2 protein measured using SPR. ( B ) Binding kinetics of SARS-CoV-2 S1 protein with ACE2 protein measured using SPR. ( C ) KKCH and SARS-CoV-2 S1 protein competition for binding to ACE2 protein measured using SPR. ( D ) The effect of KKCH on the in vitro binding of S1-RBD protein to ACE2 protein measured using ELISA. ( E ) CCK8 assay for detecting the effect of KKCH on BEAS-2B cell activity. ( F ) CCK8 assay for detecting the effect of KKCH on HEK293T cell activity. Statistically significant differences were analyzed using one-way analysis of variance (ANOVA). * p ≤ 0.05 compared with 0 μM.

Article Snippet: The ACE2 protein (no. CSB-MP866317HU) was a recombinant human protein purchased from CUSABIO (Wuhan, China).

Techniques: Binding Assay, In Vitro, Enzyme-linked Immunosorbent Assay, CCK-8 Assay, Activity Assay

Journal: Nature Communications

Article Title: Molecular insights into receptor binding energetics and neutralization of SARS-CoV-2 variants

doi: 10.1038/s41467-021-27325-1

Figure Lengend Snippet: a Schematic of a SARS-CoV-2 virus particle, expressing at its surface the spike glycoprotein (S) that mediates the binding to host cells. b The S glycoprotein is composed of two subunits, S1 and S2, and is commonly represented as a sword-like spike. The Protein Data Bank (PDB) model of this glycoprotein (ID: 6VXX) reveals how the subunits are comprised of different regions that are fundamental to the infection process. Other Structural studies (PDB ID: 6VW1) have previously obtained a complex between the receptor-binding domain (RBD, a subunit of the S glycoprotein) and the angiotensin-converting enzyme 2 (ACE2) receptor. c Featuring a 3D rendering of SARS-CoV-2, this panel showcases the key spike protein mutations in the RBD domain on each of the studied SARS-CoV-2 variants of concern: Alpha, Beta, Gamma, and Kappa. d Schematic of probing RBD mutant binding to ACE2 receptors using atomic force microscopy (AFM). RBD is covalently attached to the AFM tip via a heterobifunctional PEG-linker and their binding to ACE2 receptors immobilized on a gold (Au) coated surface is probed. Pixel-for-pixel force distance (FD) curve-based AFM approaches and retracts the tip of an AFM cantilever from the sample to record interaction forces, F , over the tip-sample distance in FD curves. e Force−time curve from which the loading rate (LR) can be extracted from the slope of the curve just before bond rupture (LR = Δ F /Δ t ) (upper curve). The contact time refers to the time when the tip and surface are in constant contact (middle curve). The lower curve shows no binding event. The tip approach is highlighted in blue, and tip retraction in red. f Representation of the system used in MD simulations.

Article Snippet: ACE2 protein (Diaclone, SAS, 715-H19-0BU) was immobilized on gold-coated surfaces using NHS-EDC chemistry.

Techniques: Virus, Expressing, Binding Assay, Infection, Mutagenesis, Microscopy

Journal: Nature Communications

Article Title: Molecular insights into receptor binding energetics and neutralization of SARS-CoV-2 variants

doi: 10.1038/s41467-021-27325-1

Figure Lengend Snippet: a Box plot of specific binding frequencies (BF) measured by AFM between the functionalized tip (RBD mutants) and the grafted ACE2 model surface or the NHS-EDC surface, lacking ACE2 receptor (dashed boxes). One data point belongs to the BF from one map acquired at 1 µm s −1 retraction speed and 250 ms contact time. The square in the box indicates the mean, the min/ max of the box the 25th and 75th percentiles respectively, and the whiskers the s.d. of the mean value. The line in the box indicates the median. N = 10 maps examined over 3 independent experiments for the ACE2 surface. N = 3 maps examined over 1–2 independent experiments for the NHS-EDC surface. P -values were determined by the two-sample t test in Origin. b – f Dynamic force spectroscopy (DFS) plot showing the individual force extracted from individual FD curves (gray data points) as well as the average rupture forces, determined at seven distinct loading rate (LR) ranges measured either between ACE2 receptor and WT-RBD ( b , N = 1434 data points), Alpha-RBD ( c , N = 1541 data points), Beta-RBD ( d , N = 1094 data points), Gamma-RBD ( e , N = 1952 data points) or Kappa-RBD ( f , N = 2719 data points). Data corresponding to single interactions were fitted with the Bell−Evans (BE) model (straight line), providing average k off and x u values. Dashed lines represent predicted binding forces for multiple simultaneous uncorrelated interactions ruptured in parallel (Williams−Evans [WE] prediction). Plots on the right: The binding frequency (BF) is plotted as a function of the contact time. Least-squares fits of the data to a mono-exponential decay curve (line) provide average kinetic on-rates ( k on ) of the probed interaction. Further calculation ( k off / k on ) leads to K D . One data point belongs to the BF from one map acquired at 1 µm s −1 retraction speed for the different contact times. All experiments were reproduced at least three times with independent tips and samples. The error bars indicate s.d. of the mean value. WT-RBD is colored in gray/black, and the VoCs Alpha-RBD in blue, Beta-RBD in green, Gamma-RBD in orange, and Kappa-RBD in purple.

Article Snippet: ACE2 protein (Diaclone, SAS, 715-H19-0BU) was immobilized on gold-coated surfaces using NHS-EDC chemistry.

Techniques: Binding Assay, Force Spectroscopy

Journal: Nature Communications

Article Title: Molecular insights into receptor binding energetics and neutralization of SARS-CoV-2 variants

doi: 10.1038/s41467-021-27325-1

Figure Lengend Snippet: a Ribbon-like representation of the RBD—ACE2 complex and highlight of residues mutated in VoCs. b Total energies and c Lennard−Jones energies for all RBD—ACE2 complexes. d Average frequencies of contacts from 7.5 μs of MD trajectory of SARS-CoV-2 WT (gray) and VoCs Alpha (blue), Beta (green), Gamma (orange), and Kappa (purple). High-frequency contacts are shown in the form (ACE2 residue number)—(RBD residue number) for seven contacts involving relevant mutations. Zoom-in on the RBD—ACE2 region around e RBD residue 501 and f around RBD residues 484 and 417 for the WT and the 4 VoCs. Sidechains are represented by sticks. Residues 38, 42, and 355 in ACE2 contacting RBD residue 501 ( e ) and residues 30, 31, 34, and 75 in ACE2 contacting RBD 417 or 484, respectively, are shown. Data in b , c show the mean and the whiskers are the s.d. of the mean value obtained from N = 3130 (WT), N = 2851 (Alpha), and N = 3130 (Beta and Kappa).

Article Snippet: ACE2 protein (Diaclone, SAS, 715-H19-0BU) was immobilized on gold-coated surfaces using NHS-EDC chemistry.

Techniques: Residue

Journal: Nature Communications

Article Title: Molecular insights into receptor binding energetics and neutralization of SARS-CoV-2 variants

doi: 10.1038/s41467-021-27325-1

Figure Lengend Snippet: Total energies ( a , c , e , g , i ) and Lennard−Jones ( b , d , f , h , j ) interaction energies between contact ACE2 and RBD residues 417 ( a , b ), 452 ( c , d ), 478 ( e , f ) 484 ( g , h ) and 501 ( i , j ). For ease of comparison, the values have been reset to zero for the WT and the initial value of the WT is shown at the top left. Each point corresponds to the mean energy and the whiskers are the s.d. around the mean value obtained from N = 3130 (WT), N = 2851 (Alpha), and N = 3130 (Beta and Kappa). For most of the data point the error bars are very small and hidden in the symbol. WT is colored in gray, and the VoCs Alpha in blue, Beta in green, Gamma in orange, and Kappa in purple.

Article Snippet: ACE2 protein (Diaclone, SAS, 715-H19-0BU) was immobilized on gold-coated surfaces using NHS-EDC chemistry.

Techniques: Comparison

Journal: Nature Communications

Article Title: Molecular insights into receptor binding energetics and neutralization of SARS-CoV-2 variants

doi: 10.1038/s41467-021-27325-1

Figure Lengend Snippet: a Ribbon-like representation of the RBD—ACE2 complex and highlights of the set of high-frequency contacts (solid green lines) that are formed by RBD conserved residues following mutations shown in black balls. b Average frequency of contacts between RBD and ACE2 molecules. These contacts are obtained from MD trajectories. Only contacts whose frequencies are larger than 0.3 and their difference to WT larger than 0.2 are shown. Bottom panels show total energies ( c , e , g , i ) and Lennard−Jones ( d , f , h , j ) interaction energies between contact ACE2 and RBD residues 475 ( c , d ), 496 ( e , f ), 498 ( g , h ) and 505 ( i , j ). Error bars in energies are given as standard deviations. Each point corresponds to the mean energy and the whiskers are the s.d. around the mean value obtained from N = 3130 (WT), N = 2851 (Alpha), and N = 3130 (Beta and Kappa). For most of the data point, the error bars are very small and hidden in the symbol. WT is colored in gray, and the VoCs Alpha in blue, Beta in green, Gamma in orange, and Kappa in purple.

Article Snippet: ACE2 protein (Diaclone, SAS, 715-H19-0BU) was immobilized on gold-coated surfaces using NHS-EDC chemistry.

Techniques:

Journal: Nature Communications

Article Title: Molecular insights into receptor binding energetics and neutralization of SARS-CoV-2 variants

doi: 10.1038/s41467-021-27325-1

Figure Lengend Snippet: a Efficiency of blocking antibodies is evaluated by measuring the binding frequency of the interaction between ACE2 and the RBD mutants before and after incubation with the three different monoclonal antibodies (mAbs) at increasing concentration (1–50 µg mL −1 ). b – f Histograms, with individual data points overlaid [ N = 3 independent experiments (tips and sample)], showing the binding frequency of the interaction between ACE2 and WT-RBD ( b ), Alpha-RBD ( c ), Beta-RBD ( d ), Gamma-RBD ( e ) or Kappa-RBD ( f ) without mAb and upon incubation with 1, 10, or 50 µg mL −1 of two monoclonal anti-RBD (B-K45 and B-R41) or a control IgG1 isotype mAb. g Graph showing the reduction of the binding frequency. Data are representative of at least N = 3 independent experiments (tips and sample) per mAb concentration. P -values were determined by two-sample t-test in Origin. The error bars indicate s.d. of the mean value. WT-RBD is colored in gray/black, and the VoCs Alpha-RBD in blue, Beta-RBD in green, Gamma-RBD in orange, and Kappa-RBD in purple.

Article Snippet: ACE2 protein (Diaclone, SAS, 715-H19-0BU) was immobilized on gold-coated surfaces using NHS-EDC chemistry.

Techniques: Blocking Assay, Binding Assay, Incubation, Bioprocessing, Concentration Assay, Control

Journal: Journal of Assisted Reproduction and Genetics

Article Title: Identification and characterization of RSIY-11, a novel seminal peptide derived from semenogelin-1, which acts as a neutral endopeptidase inhibitor modulating sperm motility

doi: 10.1007/s10815-019-01524-8

Figure Lengend Snippet: Determination of the effect of pre-incubation of RSIY-11 with NEP on inhibition. Fifty microliters of recombinant human NEP enzyme (0.1 μg/ml) and 50 μl of RSIY-11 (8.2 μM) were incubated for 0, 3, or 24 h at 37 °C prior to the addition of 50 μl of fluorogenic NEP substrate to determine enzymatic activity, and then, activity was measured every minute over 15 min. A control was performed by incubating NEP for the indicated time without RSIY-11, which was used as the 100% for each time point. Assays were performed in triplicate and averaged. Enzymatic activity was expressed as a percentage of the activity of the control. Error bars represent standard deviation. Asterisks (*) = statistically different from the control, *p value < 0.05; **p value < 0.01. NS no significant difference

Article Snippet: Michaelis-Menten Constant A fluorometric assay was performed using recombinant human NEP enzyme at 0.1 μg/ml (R&D Systems, Minneapolis, USA) and fluorogenic NEP peptide substrate ((7-methoxycoumarin-4-yl)-acetyl-R-P-P-G-F-S-A-F-K-(2, 4-dinitrophenyl)-OH, R&D Systems).

Techniques: Incubation, Inhibition, Recombinant, Activity Assay, Standard Deviation