cacl2  (New England Biolabs)


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    Structured Review

    New England Biolabs cacl2
    Cacl2, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 93 stars, based on 20 article reviews
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    Related Articles

    Immunoprecipitation:

    Article Title: The Attenuation of Trophoblast Invasion Caused by the Downregulation of EZH2 Is Involved in the Pathogenesis of Human Recurrent Miscarriage
    Article Snippet: .. Then, the mixture was lysed on ice for 10 min. After addition of 500 μL of MNase buffer (85 mM Tris-HCl [pH 7.5], 3 mM MgCl2, 2 mM CaCl2, and 0.3 M sucrose) containing 2 μg/mL BSA and 4000 U/mL Micrococcal Nuclease (M0247; NEB) per immunoprecipitation (IP) prep, the nuclei were then subjected to digestion for 10 min at 37°C, with frequent inversion every 3–5 min. .. The digestion was stopped by the addition of EDTA to a final concentration of 20 mM.

    Size-exclusion Chromatography:

    Article Title: TRF2 is recruited to the pre-initiation complex as a testis-specific subunit of TFIIA/ALF to promote haploid cell gene expression
    Article Snippet: .. The lysate was sonicated for 30 sec as described above, CaCl2 and MgCl2 were added to 5 mM final concentration each and digestion was performed with MNase (10 gel units/uL final) (M0247S, New England BioLabs) for 10 min of incubation. .. The reaction was stopped by addition of EDTA to 10 mM final concentration.

    Activation Assay:

    Article Title: Development of a specific affinity-matured exosite inhibitor to MT1-MMP that efficiently inhibits tumor cell invasion in vitro and metastasis in vivo
    Article Snippet: .. Pro-MT1-MMP activation assay Pro-MT1-MMP E240A (2 μM) was preincubated with Fc-scFv's (3 μM) for 30 minutes, RT, in 100 mM HEPES, 10 mM CaCl2 , 0.5% triton X-100, before 1 unit of furin (NEB) was added. .. After 3 hours at 30°C, products were analyzed by non-reducing SDS-PAGE.

    Concentration Assay:

    Article Title: TRF2 is recruited to the pre-initiation complex as a testis-specific subunit of TFIIA/ALF to promote haploid cell gene expression
    Article Snippet: .. The lysate was sonicated for 30 sec as described above, CaCl2 and MgCl2 were added to 5 mM final concentration each and digestion was performed with MNase (10 gel units/uL final) (M0247S, New England BioLabs) for 10 min of incubation. .. The reaction was stopped by addition of EDTA to 10 mM final concentration.

    Incubation:

    Article Title: A novel galactolipase from a green microalga Chlorella kessleri: purification, characterization, molecular cloning, and heterologous expression
    Article Snippet: .. The purified His-tag fused TF-ckGL sample was incubated at 7 °C for 24 h in 2 mM CaCl2 , 0.1 M NaCl/50 mM buffer A, and 3 mU factor Xa (New England Biolabs Japan Inc., Tokyo), after which it was applied to a HisTrap HP column (1 mL) equilibrated with 5 mM imidazole and 0.5 M NaCl/buffer T (pH 7.5). ..

    Article Title: Time-dependent increase in ribosome processivity
    Article Snippet: .. Lysate was nucleased by incubation at 18°C for 5 min in the presence of 0.62 mM CaCl2 and 2500 gel units/ml micrococcal nuclease (New England Biolabs). ..

    Article Title: TRF2 is recruited to the pre-initiation complex as a testis-specific subunit of TFIIA/ALF to promote haploid cell gene expression
    Article Snippet: .. The lysate was sonicated for 30 sec as described above, CaCl2 and MgCl2 were added to 5 mM final concentration each and digestion was performed with MNase (10 gel units/uL final) (M0247S, New England BioLabs) for 10 min of incubation. .. The reaction was stopped by addition of EDTA to 10 mM final concentration.

    Sonication:

    Article Title: TRF2 is recruited to the pre-initiation complex as a testis-specific subunit of TFIIA/ALF to promote haploid cell gene expression
    Article Snippet: .. The lysate was sonicated for 30 sec as described above, CaCl2 and MgCl2 were added to 5 mM final concentration each and digestion was performed with MNase (10 gel units/uL final) (M0247S, New England BioLabs) for 10 min of incubation. .. The reaction was stopped by addition of EDTA to 10 mM final concentration.

    Purification:

    Article Title: A novel galactolipase from a green microalga Chlorella kessleri: purification, characterization, molecular cloning, and heterologous expression
    Article Snippet: .. The purified His-tag fused TF-ckGL sample was incubated at 7 °C for 24 h in 2 mM CaCl2 , 0.1 M NaCl/50 mM buffer A, and 3 mU factor Xa (New England Biolabs Japan Inc., Tokyo), after which it was applied to a HisTrap HP column (1 mL) equilibrated with 5 mM imidazole and 0.5 M NaCl/buffer T (pH 7.5). ..

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    New England Biolabs dnase i
    Rate of transfer of DNA from UvrA to UvrB <t>DNase</t> I footprinting of Bca UvrA and Bca UvrB-DNA complexes. Reaction mixtures contained UvrA 2 (10 nM), ± 100 nM UvrB, 2 nM F 26 50/NDB duplex with the radiolabel on the damaged strand. The reactions were incubated at 37 °C then DNase I was added for 30 sec at RT. Then the samples were processed as described in the methods. The time indicated in the figure includes the time for DNase I digestion. Panel a , Gel image showing the transition of the WT UvrA footprint to WT UvrB footprint. The right side of the gel contains a graphic representation of the DNase I footprints observed. Panel b , Gel image showing the transition of the KRAA UvrA footprint to the WT UvrB footprint. The position of the adduct and the 3′ and 5′ incision sites are noted on the left-hand side of the gels. Asterisks indicate the position of the band used to quantify the gels, p1. Panel c , Graphic representation of the band intensities of p1 relative to the band intensity observed in lane 4, DNase I digestion in the absence of proteins. The average of 3 independent experiments was plotted with the standard deviation at each point. Data were fit to a single or double exponential using Excel.
    Dnase I, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    New England Biolabs mnase reaction buffer
    Characterization of the NRS sequences. ( A ) EMSA comparing binding of the <t>DNA</t> binding domain of GR to a GBS sequence flanked by either the control sequence (left) or by NRS2 (right). ( B ) DNase I accessibility assay was performed with populations of transgenic cells with stably integrated reporters as indicated. Regions of interest were analyzed by qPCR ( FKBP5 : control accessible region; IGFBP1 : control inaccessible region; integr. GBS: integrated reporter region). Results are shown as % of input remaining after DNAse I digestion ±SEM (n = 3). ( C ) Nucleosome occupancy was analyzed using micrococcal nuclease <t>(MNase)</t> assays for populations of transgenic cells with stably integrated reporters as indicated. Regions of interest were analyzed by qPCR. Integr. GBS: integrated reporter region. Results are shown as % of input remaining after MNase digestion ±SEM (n = 3).
    Mnase Reaction Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs heparinase reaction buffer
    Effect of heparin sodium salt, sodium chlorate and <t>heparinase</t> II treatment in binding assay. (a) Western blotting analysis following treatment with heparin sodium salt. Relative intensity of immunofluorescence signal derived from different binding assays (b) heparin sodium salt, (c) sodium chlorate and (d) heparinase II. Mean fluorescence intensity of stained cells was measured with ImageJ. Error bars represent standard deviation. *, p
    Heparinase Reaction Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    New England Biolabs furin cleavage buffer
    (A) Myc-L2-HA HPV16 PsV is sensitive to <t>HD5.</t> HeLa cells were infected with WT HPV16 PsV in SFM (black circles, IC 50 = 1.47 μM, 95% CI = 1.35 to 1.6 μM), Myc-L2-HA PsV in SFM (open circles, IC 50 = 1.71 μM, 95% CI = 1.6 to 1.8 μM), or 10 times as much Myc-L2-HA PsV in complete medium (gray circles, IC 50 = 0.79 μM, 95% CI = 0.69 to 0.9 μM) incubated with increasing concentrations of HD5. Data are means ± SD from three independent experiments normalized to infection in the absence of inhibitor. (B) HD5 inhibits <t>furin</t> cleavage of L2. HeLa cells were infected with Myc-16L2-HA HPV16 PsV in the presence of 40 μM furin inhibitor (FI), 1 to 10 μM HD5, or no inhibitor (−). L2 cleavage was assessed by immunoblotting of cell lysates 16 h p.i. using an anti-HA antibody. Cleaved L2 (arrow) is visible as a faster-migrating band below uncleaved L2. Shown are three independent experiments. (C) Analysis of a greater amount of lysate confirms the inhibition of furin cleavage. Different amounts of HD5-treated HPV16 PsV lysate, indicated by fold change relative to the amounts loaded in panel B, were assessed by immunoblotting with anti-HA antibody. (D) HD5 does not directly affect the enzymatic activity of furin. A total of 1.8 ng rL2:1–160 was digested with 1 U of furin in the presence or absence of the indicated inhibitors for 1 h at 30°C. Samples were immunoblotted using an anti-His antibody. Cleaved rL2:1–160 is the faster-migrating band. Shown are two independent experiments. (E) Titration of furin required for rL2:1–160 cleavage. rL2:1–160 was digested with a 3-fold dilution series of furin, starting at 1 U of total furin. Samples were resolved on a reducing gel and immunoblotted using anti-His antibody. Cleaved rL2:1–160 is the faster-migrating band.
    Furin Cleavage Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Rate of transfer of DNA from UvrA to UvrB DNase I footprinting of Bca UvrA and Bca UvrB-DNA complexes. Reaction mixtures contained UvrA 2 (10 nM), ± 100 nM UvrB, 2 nM F 26 50/NDB duplex with the radiolabel on the damaged strand. The reactions were incubated at 37 °C then DNase I was added for 30 sec at RT. Then the samples were processed as described in the methods. The time indicated in the figure includes the time for DNase I digestion. Panel a , Gel image showing the transition of the WT UvrA footprint to WT UvrB footprint. The right side of the gel contains a graphic representation of the DNase I footprints observed. Panel b , Gel image showing the transition of the KRAA UvrA footprint to the WT UvrB footprint. The position of the adduct and the 3′ and 5′ incision sites are noted on the left-hand side of the gels. Asterisks indicate the position of the band used to quantify the gels, p1. Panel c , Graphic representation of the band intensities of p1 relative to the band intensity observed in lane 4, DNase I digestion in the absence of proteins. The average of 3 independent experiments was plotted with the standard deviation at each point. Data were fit to a single or double exponential using Excel.

    Journal: DNA repair

    Article Title: Cooperative damage recognition by UvrA and UvrB: Identification of UvrA residues that mediate DNA binding

    doi: 10.1016/j.dnarep.2007.11.013

    Figure Lengend Snippet: Rate of transfer of DNA from UvrA to UvrB DNase I footprinting of Bca UvrA and Bca UvrB-DNA complexes. Reaction mixtures contained UvrA 2 (10 nM), ± 100 nM UvrB, 2 nM F 26 50/NDB duplex with the radiolabel on the damaged strand. The reactions were incubated at 37 °C then DNase I was added for 30 sec at RT. Then the samples were processed as described in the methods. The time indicated in the figure includes the time for DNase I digestion. Panel a , Gel image showing the transition of the WT UvrA footprint to WT UvrB footprint. The right side of the gel contains a graphic representation of the DNase I footprints observed. Panel b , Gel image showing the transition of the KRAA UvrA footprint to the WT UvrB footprint. The position of the adduct and the 3′ and 5′ incision sites are noted on the left-hand side of the gels. Asterisks indicate the position of the band used to quantify the gels, p1. Panel c , Graphic representation of the band intensities of p1 relative to the band intensity observed in lane 4, DNase I digestion in the absence of proteins. The average of 3 independent experiments was plotted with the standard deviation at each point. Data were fit to a single or double exponential using Excel.

    Article Snippet: After incubation at 37 °C for the indicated amount of time, DNase I (1 µL of 0.03 U/µL in 50 mM CaCl2 and 100 nM bovine serum albumin, New England BioLabs) was added and after digestion for 30 sec at room temperature the reactions were stopped by the addition of sarkosyl (0.5%) and EDTA (15 mM).

    Techniques: Footprinting, BIA-KA, Incubation, Size-exclusion Chromatography, Standard Deviation

    DNase I Footprinting DNase I footprint of the WT and KRAA UvrA proteins bound to the 50 bp F 26 50/NBD duplex with the 32 P on the 5′ end of the damaged strand. The indicated proteins were incubated with 2 nM duplex in the presence of 50 mM Tris-HCl, pH 7.5, 50 mM KCl, 10 MgCl 2 , 1 mM ATP, 10 mM DTT, 100 nM bovine serum albumin for 15 min at 37 °C then DNase I treated and processed as described in the methods. Panel a , WT UvrA DNase I footprint. Panel b , KRAA UvrA DNase I footprint. Both gels are loaded in the same order. The lanes contained the following: lane 1, no protein and no DNase I; lane 2, UvrA 2 , 10 nM and no DNase I; lane 3, no protein plus DNase I; lanes 4–7, increasing concentrations of UvrA 2 , 10 – 80 nM, plus DNase I; lane 8, UvrA 2 , 10 nM, WT UvrB, 100 nM, plus DNase I; lane 9, WT UvrB, 100 nM, plus DNase I. The position of the adduct and the 3′ and 5′ incision sites are noted on the left-hand side of the gels. The bands that were quantified for the graph in panel c are denoted by the arrows, p1 and p2. Panel c ]. For more details see the Materials and Methods.

    Journal: DNA repair

    Article Title: Cooperative damage recognition by UvrA and UvrB: Identification of UvrA residues that mediate DNA binding

    doi: 10.1016/j.dnarep.2007.11.013

    Figure Lengend Snippet: DNase I Footprinting DNase I footprint of the WT and KRAA UvrA proteins bound to the 50 bp F 26 50/NBD duplex with the 32 P on the 5′ end of the damaged strand. The indicated proteins were incubated with 2 nM duplex in the presence of 50 mM Tris-HCl, pH 7.5, 50 mM KCl, 10 MgCl 2 , 1 mM ATP, 10 mM DTT, 100 nM bovine serum albumin for 15 min at 37 °C then DNase I treated and processed as described in the methods. Panel a , WT UvrA DNase I footprint. Panel b , KRAA UvrA DNase I footprint. Both gels are loaded in the same order. The lanes contained the following: lane 1, no protein and no DNase I; lane 2, UvrA 2 , 10 nM and no DNase I; lane 3, no protein plus DNase I; lanes 4–7, increasing concentrations of UvrA 2 , 10 – 80 nM, plus DNase I; lane 8, UvrA 2 , 10 nM, WT UvrB, 100 nM, plus DNase I; lane 9, WT UvrB, 100 nM, plus DNase I. The position of the adduct and the 3′ and 5′ incision sites are noted on the left-hand side of the gels. The bands that were quantified for the graph in panel c are denoted by the arrows, p1 and p2. Panel c ]. For more details see the Materials and Methods.

    Article Snippet: After incubation at 37 °C for the indicated amount of time, DNase I (1 µL of 0.03 U/µL in 50 mM CaCl2 and 100 nM bovine serum albumin, New England BioLabs) was added and after digestion for 30 sec at room temperature the reactions were stopped by the addition of sarkosyl (0.5%) and EDTA (15 mM).

    Techniques: Footprinting, Incubation

    Characterization of the NRS sequences. ( A ) EMSA comparing binding of the DNA binding domain of GR to a GBS sequence flanked by either the control sequence (left) or by NRS2 (right). ( B ) DNase I accessibility assay was performed with populations of transgenic cells with stably integrated reporters as indicated. Regions of interest were analyzed by qPCR ( FKBP5 : control accessible region; IGFBP1 : control inaccessible region; integr. GBS: integrated reporter region). Results are shown as % of input remaining after DNAse I digestion ±SEM (n = 3). ( C ) Nucleosome occupancy was analyzed using micrococcal nuclease (MNase) assays for populations of transgenic cells with stably integrated reporters as indicated. Regions of interest were analyzed by qPCR. Integr. GBS: integrated reporter region. Results are shown as % of input remaining after MNase digestion ±SEM (n = 3).

    Journal: Nucleic Acids Research

    Article Title: Identification and characterization of DNA sequences that prevent glucocorticoid receptor binding to nearby response elements

    doi: 10.1093/nar/gkw203

    Figure Lengend Snippet: Characterization of the NRS sequences. ( A ) EMSA comparing binding of the DNA binding domain of GR to a GBS sequence flanked by either the control sequence (left) or by NRS2 (right). ( B ) DNase I accessibility assay was performed with populations of transgenic cells with stably integrated reporters as indicated. Regions of interest were analyzed by qPCR ( FKBP5 : control accessible region; IGFBP1 : control inaccessible region; integr. GBS: integrated reporter region). Results are shown as % of input remaining after DNAse I digestion ±SEM (n = 3). ( C ) Nucleosome occupancy was analyzed using micrococcal nuclease (MNase) assays for populations of transgenic cells with stably integrated reporters as indicated. Regions of interest were analyzed by qPCR. Integr. GBS: integrated reporter region. Results are shown as % of input remaining after MNase digestion ±SEM (n = 3).

    Article Snippet: For each sample containing 0.8 μg genomic DNA in 50 μl storage buffer, an equal volume of MNase reaction buffer (50 mM Tris pH 7.4; 25 mM KCl; 2.5 mM CaCl2 ; 5 mM MgCl2 ; 12.5 % glycerol) was added containing 1 μl MNase (NEB; ∼200 kunitz) for MNase-treated samples.

    Techniques: Binding Assay, Sequencing, Transgenic Assay, Stable Transfection, Real-time Polymerase Chain Reaction

    Effect of heparin sodium salt, sodium chlorate and heparinase II treatment in binding assay. (a) Western blotting analysis following treatment with heparin sodium salt. Relative intensity of immunofluorescence signal derived from different binding assays (b) heparin sodium salt, (c) sodium chlorate and (d) heparinase II. Mean fluorescence intensity of stained cells was measured with ImageJ. Error bars represent standard deviation. *, p

    Journal: PLoS ONE

    Article Title: C-Terminal Amino Acids 471-507 of Avian Hepatitis E Virus Capsid Protein Are Crucial for Binding to Avian and Human Cells

    doi: 10.1371/journal.pone.0153723

    Figure Lengend Snippet: Effect of heparin sodium salt, sodium chlorate and heparinase II treatment in binding assay. (a) Western blotting analysis following treatment with heparin sodium salt. Relative intensity of immunofluorescence signal derived from different binding assays (b) heparin sodium salt, (c) sodium chlorate and (d) heparinase II. Mean fluorescence intensity of stained cells was measured with ImageJ. Error bars represent standard deviation. *, p

    Article Snippet: Heparinase II treatment of LMH cells LMH cells were pre-treated in Heparinase Reaction Buffer (20 mM Tris-HCl, 100 mM NaCl and 1.5 mM CaCl2 ) with heparinase II (10U/ml) from Bacteroides (New England Biolabs GmbH, Frankfurt am Main, Germany) or PBS as control for 2h at 37°C in an atmosphere supplied with 5% CO2 .

    Techniques: Binding Assay, Western Blot, Immunofluorescence, Derivative Assay, Fluorescence, Staining, Standard Deviation

    (A) Myc-L2-HA HPV16 PsV is sensitive to HD5. HeLa cells were infected with WT HPV16 PsV in SFM (black circles, IC 50 = 1.47 μM, 95% CI = 1.35 to 1.6 μM), Myc-L2-HA PsV in SFM (open circles, IC 50 = 1.71 μM, 95% CI = 1.6 to 1.8 μM), or 10 times as much Myc-L2-HA PsV in complete medium (gray circles, IC 50 = 0.79 μM, 95% CI = 0.69 to 0.9 μM) incubated with increasing concentrations of HD5. Data are means ± SD from three independent experiments normalized to infection in the absence of inhibitor. (B) HD5 inhibits furin cleavage of L2. HeLa cells were infected with Myc-16L2-HA HPV16 PsV in the presence of 40 μM furin inhibitor (FI), 1 to 10 μM HD5, or no inhibitor (−). L2 cleavage was assessed by immunoblotting of cell lysates 16 h p.i. using an anti-HA antibody. Cleaved L2 (arrow) is visible as a faster-migrating band below uncleaved L2. Shown are three independent experiments. (C) Analysis of a greater amount of lysate confirms the inhibition of furin cleavage. Different amounts of HD5-treated HPV16 PsV lysate, indicated by fold change relative to the amounts loaded in panel B, were assessed by immunoblotting with anti-HA antibody. (D) HD5 does not directly affect the enzymatic activity of furin. A total of 1.8 ng rL2:1–160 was digested with 1 U of furin in the presence or absence of the indicated inhibitors for 1 h at 30°C. Samples were immunoblotted using an anti-His antibody. Cleaved rL2:1–160 is the faster-migrating band. Shown are two independent experiments. (E) Titration of furin required for rL2:1–160 cleavage. rL2:1–160 was digested with a 3-fold dilution series of furin, starting at 1 U of total furin. Samples were resolved on a reducing gel and immunoblotted using anti-His antibody. Cleaved rL2:1–160 is the faster-migrating band.

    Journal: Journal of Virology

    Article Title: Alpha-Defensin HD5 Inhibits Furin Cleavage of Human Papillomavirus 16 L2 To Block Infection

    doi: 10.1128/JVI.02901-14

    Figure Lengend Snippet: (A) Myc-L2-HA HPV16 PsV is sensitive to HD5. HeLa cells were infected with WT HPV16 PsV in SFM (black circles, IC 50 = 1.47 μM, 95% CI = 1.35 to 1.6 μM), Myc-L2-HA PsV in SFM (open circles, IC 50 = 1.71 μM, 95% CI = 1.6 to 1.8 μM), or 10 times as much Myc-L2-HA PsV in complete medium (gray circles, IC 50 = 0.79 μM, 95% CI = 0.69 to 0.9 μM) incubated with increasing concentrations of HD5. Data are means ± SD from three independent experiments normalized to infection in the absence of inhibitor. (B) HD5 inhibits furin cleavage of L2. HeLa cells were infected with Myc-16L2-HA HPV16 PsV in the presence of 40 μM furin inhibitor (FI), 1 to 10 μM HD5, or no inhibitor (−). L2 cleavage was assessed by immunoblotting of cell lysates 16 h p.i. using an anti-HA antibody. Cleaved L2 (arrow) is visible as a faster-migrating band below uncleaved L2. Shown are three independent experiments. (C) Analysis of a greater amount of lysate confirms the inhibition of furin cleavage. Different amounts of HD5-treated HPV16 PsV lysate, indicated by fold change relative to the amounts loaded in panel B, were assessed by immunoblotting with anti-HA antibody. (D) HD5 does not directly affect the enzymatic activity of furin. A total of 1.8 ng rL2:1–160 was digested with 1 U of furin in the presence or absence of the indicated inhibitors for 1 h at 30°C. Samples were immunoblotted using an anti-His antibody. Cleaved rL2:1–160 is the faster-migrating band. Shown are two independent experiments. (E) Titration of furin required for rL2:1–160 cleavage. rL2:1–160 was digested with a 3-fold dilution series of furin, starting at 1 U of total furin. Samples were resolved on a reducing gel and immunoblotted using anti-His antibody. Cleaved rL2:1–160 is the faster-migrating band.

    Article Snippet: A total of 1.8 ng of rL2:1–160 was incubated with or without 1, 5, or 10 μM HD5 or 40 μM furin inhibitor in 20 μl furin cleavage buffer (100 mM HEPES-1 mM CaCl2 , pH 7.4) on ice for 45 min. One unit of furin (NEB) was added, and the samples were incubated at 30°C for 1 h. Reduced, denatured samples were resolved on 15% SDS-PAGE gels and analyzed by immunoblotting with mouse anti-His antibody (1:1,000; Thermo-Fisher) and goat anti-mouse HRP (1:5,000; Thermo-Fisher).

    Techniques: Infection, Incubation, Inhibition, Activity Assay, Titration