A 3 ml volume of pseudoparticles was pelleted using a TLA-55 rotor with an Optima-MAX-E ultracentrifuge (Beckman Coulter) for 2 hours at 42,000 rpm at 4°C. untreated particles were resuspended in 30 μl DPBS buffer. Pseudopartices were generated as described in the pseudoparticle generation section, with the furin inhibitor dec-RVKR-CMK (Cat:35–011, Tocris) being added during transfection to select wells. For the + furin treated MLVpps, particles were resuspended in 30 μL of furin buffer consistent in 20 mM HEPES, 0.2 mM CaCl2, and 0.2 mM β-mercaptoethanol (at pH 7.0). Furin-treated particles were later incubated with 6 U of recombinant furin for 3 h at 37 °C. Sodium dodecyl sulfate (SDS) loading buffer and DTT were added to all samples and heated at 95°C for 10 minutes. Samples were separated on NuPAGE Bis-Tris gel (Invitrogen) and transferred on polyvinylidene difluoride membranes (GE). SARS-CoV-2 S was detected using a rabbit polyclonal antibody against the S2 domain (Cat: 40590-T62, Sinobiological) and an AlexaFluor 488 goat anti-rabbit antibody. Bands were detected using the ChemiDoc Imaging software (Bio-Rad) and band intensity was calculated using the analysis tools on Biorad Image Lab 6.1 software to determine the uncleaved to cleaved S ratios.
Dec rvkr cmk
Manufactured by Bio-Techne
Sourced in United Kingdom
Dec-RVKR-CMK is a chemical compound that acts as a protease inhibitor. It functions by blocking the activity of specific proteases, enzymes responsible for cleaving peptide bonds in proteins.
Automatically generated - may contain errors
Lab products found in correlation
Polyvinylidene difluoride membrane, by GE Healthcare (3 mentions)
Optima max e, by Beckman Coulter (3 mentions)
Chemidoc imaging software, by Bio-Rad (3 mentions)
Image lab 6, by Bio-Rad (3 mentions)
Nupage bis tris gel, by Thermo Fisher Scientific (3 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
X complete protease inhibitor cocktail, by Roche (1 mentions)
V5 antibody, by Thermo Fisher Scientific (1 mentions)
5 protocols using dec rvkr cmk
1
SARS-CoV-2 Spike Protein Cleavage Assay
2
SARS-CoV-2 Inhibition by Protease Inhibitor
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Calu3 or VeroE6 cells were infected with SARS-CoV-2 for 1 h and were treated with dec-RVKR-cmk (3501, Tocris Bioscience, Bristol, United Kingdom) at 4 μM or 20 μM, or were mock-treated. At 24hpi, the cell lysates and supernatants were harvested for qRT-PCR quantification of virus replication. In parallel, cell lysates were harvested at 72hpi for western blot detection of spike cleavage.
3
Quantification of SARS-CoV-2 Spike Protein Cleavage
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A 3-mL volume of pseudoparticles was pelleted using a TLA-55 rotor with an Optima-MAX-E ultracentrifuge (Beckman Coulter) for 2 h at 42,000 rpm at 4°C. Untreated particles were resuspended in 30 μL DPBS buffer. Pseudoparticles were generated as described in the pseudoparticle generation section, with the furin inhibitor dec-RVKR-CMK (cat.: 35-011, Tocris) being added during transfection to select wells. For the + furin treated MLVpps, particles were resuspended in 30 μL of furin buffer consistent in 20 mM HEPES, 0.2 mM CaCl2, and 0.2 mM β-mercaptoethanol (at pH 7.0). Furin-treated particles were later incubated with 6 U of recombinant furin for 3 h at 37°C. Sodium dodecyl sulfate (SDS) loading buffer and DTT were added to all samples and heated at 95°C for 10 min. Samples were separated on NuPAGE Bis-Tris gel (Invitrogen) and transferred on polyvinylidene difluoride membranes (GE). SARS-CoV-2 S was detected using a rabbit polyclonal antibody against the S2 domain (cat.: 40590-T62, Sinobiological). Bands were detected using the ChemiDoc Imaging software (Bio-Rad), and band intensity was calculated using the analysis tools on Bio-Rad Image Lab 6.1 software to determine the uncleaved to cleaved S ratios.
4
Quantification of SARS-CoV-2 Spike Protein Cleavage
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+ Expand
A 3-mL volume of pseudoparticles was pelleted using a TLA-55 rotor with an Optima-MAX-E ultracentrifuge (Beckman Coulter) for 2 h at 42,000 rpm at 4°C. Untreated particles were resuspended in 30 μL DPBS buffer. Pseudoparticles were generated as described in the pseudoparticle generation section, with the furin inhibitor dec-RVKR-CMK (cat.: 35-011, Tocris) being added during transfection to select wells. For the + furin treated MLVpps, particles were resuspended in 30 μL of furin buffer consistent in 20 mM HEPES, 0.2 mM CaCl2, and 0.2 mM β-mercaptoethanol (at pH 7.0). Furin-treated particles were later incubated with 6 U of recombinant furin for 3 h at 37°C. Sodium dodecyl sulfate (SDS) loading buffer and DTT were added to all samples and heated at 95°C for 10 min. Samples were separated on NuPAGE Bis-Tris gel (Invitrogen) and transferred on polyvinylidene difluoride membranes (GE). SARS-CoV-2 S was detected using a rabbit polyclonal antibody against the S2 domain (cat.: 40590-T62, Sinobiological). Bands were detected using the ChemiDoc Imaging software (Bio-Rad), and band intensity was calculated using the analysis tools on Bio-Rad Image Lab 6.1 software to determine the uncleaved to cleaved S ratios.
5
Profiling Furin and PC Enzyme Activities on FGF23 Processing
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The cDNAs coding for human furin, PC7, and PACE4, and mouse PC5A and PC5B were cloned into the pIRES2-EGFP vector (31 (link)), while the cDNA of hFGF23 was cloned in pIRES2-EGFP-V5. CHO-K1 and furin-deficient CHO-FD11 cells were cultured at 37°C and 5% CO2 in Ham’s F-12 medium supplemented with 10% (v/v) FBS (32 (link)) and transfected with 3 µg of plasmid following the standard protocol for Lipofectamine 2000 (Thermo Fisher). For the PCs inhibition, CHO-K1 cells were transfected with cDNAs encoding for hFGF23-V5 and PCs in the ratio of 5:1 and 24 hours later pretreated for 5 hours with Dec-RVKR-CMK (50 μM; Tocris) or D6R (20 μM; Calbiochem) followed by 22 hours of treatment in serum free media. Cells supernatant was then collected in the presence of 1x complete protease inhibitor cocktail (Roche) and FGF23 processing was analyzed by SDS-PAGE (15% Tris-glycine) followed by western blot using V5 antibody (Thermo Fisher).
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