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  • 99
    Worthington Biochemical trypsin
    Trypsin, supplied by Worthington Biochemical, used in various techniques. Bioz Stars score: 99/100, based on 2202 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore 1x trypsin
    1x Trypsin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Bioneer Corporation trypure
    Trypure, supplied by Bioneer Corporation, used in various techniques. Bioz Stars score: 93/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore trypsin
    Trypsin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 19492 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore trypsin solution
    Trypsin Solution, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 92 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore trypsin edta
    Inhibition of <t>KSHV</t> infection by anti-integrin antibodies and soluble integrins. (A) Inhibition of GFP-KSHV infection by anti-integrin antibodies. Monolayers of HMVEC-d and HFF were incubated with 10 μg/ml of MAbs against integrins or integrin subunits for 1 h at 4°C, washed, and infected with GFP-KSHV at an MOI of 5 DNA copies/cell. After incubation for 2 h at 37°C, the cells were washed and incubated for 72 h, and the number of GFP-positive cells was estimated under a fluorescence microscope. Each experiment was done in duplicate, and each bar represents the average plus the standard deviation (SD) of three experiments. (B) Effects of anti-integrin antibodies on KSHV binding. HFF were incubated with anti-integrin antibodies (10 μg/ml) at 4°C for 90 min and incubated with a predetermined concentration of [ 3 H]thymidine-labeled purified KSHV. The labeled KSHV was also mixed with 10 μg/ml of heparin or soluble integrins for 90 min at 4°C and then added to the cells. After incubation for 90 min at 4°C with the virus, the cells were washed, lysed, precipitated with trichloroacetic acid, and counted. The bound virus cpm in the absence of any treatment was considered to be 100% and was used to calculate the percentage inhibition of virus binding. Each reaction was done in triplicate, and each bar represents the average plus the SD of three independent experiments. The results with HFF are shown. (C) Inhibition of KSHV infection by soluble integrins. KSHV (5 DNA copies/cell) was mixed with 10 μg/ml of heparin or 10 μg/ml soluble integrins for 2 h at 37°C and added to the cells. After 2 h, the cells were washed to remove the unbound virus, treated with 0.25% <t>trypsin-EDTA</t> for 5 min at 37°C to remove the bound but noninternalized viruses, and washed, and total DNA was isolated. The number of KSHV ORF73 copies was estimated by real-time DNA PCR. The cycle threshold values were used to plot the standard graph and to calculate the relative copy numbers of viral DNA in the samples. The data are presented as percentages of inhibition of KSHV DNA internalization obtained when the cells were incubated with the virus alone. Each reaction was done in duplicate, and each bar represents the average ± SD of three experiments. (D) KSHV was mixed with 10 μg/ml soluble integrin combinations for 2 h at 37°C and added to HFF. After 2 h, the cells were washed to remove the unbound virus and treated with 0.25% trypsin-EDTA for 5 min at 37°C to remove the bound but noninternalized viruses, and the internalized viral DNA was estimated by real-time DNA PCR. Each reaction was done in duplicate, and each bar represents the average ± the SD of three experiments.
    Trypsin Edta, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 7480 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore 1x trypsin edta
    Inhibition of <t>KSHV</t> infection by anti-integrin antibodies and soluble integrins. (A) Inhibition of GFP-KSHV infection by anti-integrin antibodies. Monolayers of HMVEC-d and HFF were incubated with 10 μg/ml of MAbs against integrins or integrin subunits for 1 h at 4°C, washed, and infected with GFP-KSHV at an MOI of 5 DNA copies/cell. After incubation for 2 h at 37°C, the cells were washed and incubated for 72 h, and the number of GFP-positive cells was estimated under a fluorescence microscope. Each experiment was done in duplicate, and each bar represents the average plus the standard deviation (SD) of three experiments. (B) Effects of anti-integrin antibodies on KSHV binding. HFF were incubated with anti-integrin antibodies (10 μg/ml) at 4°C for 90 min and incubated with a predetermined concentration of [ 3 H]thymidine-labeled purified KSHV. The labeled KSHV was also mixed with 10 μg/ml of heparin or soluble integrins for 90 min at 4°C and then added to the cells. After incubation for 90 min at 4°C with the virus, the cells were washed, lysed, precipitated with trichloroacetic acid, and counted. The bound virus cpm in the absence of any treatment was considered to be 100% and was used to calculate the percentage inhibition of virus binding. Each reaction was done in triplicate, and each bar represents the average plus the SD of three independent experiments. The results with HFF are shown. (C) Inhibition of KSHV infection by soluble integrins. KSHV (5 DNA copies/cell) was mixed with 10 μg/ml of heparin or 10 μg/ml soluble integrins for 2 h at 37°C and added to the cells. After 2 h, the cells were washed to remove the unbound virus, treated with 0.25% <t>trypsin-EDTA</t> for 5 min at 37°C to remove the bound but noninternalized viruses, and washed, and total DNA was isolated. The number of KSHV ORF73 copies was estimated by real-time DNA PCR. The cycle threshold values were used to plot the standard graph and to calculate the relative copy numbers of viral DNA in the samples. The data are presented as percentages of inhibition of KSHV DNA internalization obtained when the cells were incubated with the virus alone. Each reaction was done in duplicate, and each bar represents the average ± SD of three experiments. (D) KSHV was mixed with 10 μg/ml soluble integrin combinations for 2 h at 37°C and added to HFF. After 2 h, the cells were washed to remove the unbound virus and treated with 0.25% trypsin-EDTA for 5 min at 37°C to remove the bound but noninternalized viruses, and the internalized viral DNA was estimated by real-time DNA PCR. Each reaction was done in duplicate, and each bar represents the average ± the SD of three experiments.
    1x Trypsin Edta, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    Millipore trypsin inhibitor
    The HPLC elution patterns of <t>Kunitz</t> trypsin inhibitor (KTI), <t>lectin</t> and soybean whey.
    Trypsin Inhibitor, supplied by Millipore, used in various techniques. Bioz Stars score: 98/100, based on 4489 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore trypsin powder
    The HPLC elution patterns of <t>Kunitz</t> trypsin inhibitor (KTI), <t>lectin</t> and soybean whey.
    Trypsin Powder, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore trypsin edta 1x
    The HPLC elution patterns of <t>Kunitz</t> trypsin inhibitor (KTI), <t>lectin</t> and soybean whey.
    Trypsin Edta 1x, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 30 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore crystallized trypsin
    The HPLC elution patterns of <t>Kunitz</t> trypsin inhibitor (KTI), <t>lectin</t> and soybean whey.
    Crystallized Trypsin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare trypsin
    The HPLC elution patterns of <t>Kunitz</t> trypsin inhibitor (KTI), <t>lectin</t> and soybean whey.
    Trypsin, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 1332 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Lonza trypsin
    The HPLC elution patterns of <t>Kunitz</t> trypsin inhibitor (KTI), <t>lectin</t> and soybean whey.
    Trypsin, supplied by Lonza, used in various techniques. Bioz Stars score: 99/100, based on 612 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore trypsin singles sigma
    The HPLC elution patterns of <t>Kunitz</t> trypsin inhibitor (KTI), <t>lectin</t> and soybean whey.
    Trypsin Singles Sigma, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore pancreatic trypsin
    The HPLC elution patterns of <t>Kunitz</t> trypsin inhibitor (KTI), <t>lectin</t> and soybean whey.
    Pancreatic Trypsin, supplied by Millipore, 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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    99
    Millipore proteomics grade trypsin
    Detection of clinically relevant natural protein variants in the PREFER plasma proteomes. Mass spectrometry-based <t>proteomics</t> data was reanalyzed using a modified plasma-specific protein database containing all known forms of naturally occurring clinically-relevant variants. (a) Bar plot for each protein variant where the y -axis shows the summed intensity of peptides derived from each allele as a percentage of total, and the x -axis shows the 44 participant samples with one bar per sample. The gene name for each variant and its position in the protein are indicated in bold to the right of each plot. Also shown are the associated human phenotypes. Peptides from each allele are shown in different colors on the same plot and correspond to each legend shown on the right. (b) Bar plot showing either the frequency of the genotype in the European population sampled by the 1000Genomes study, or the frequency of the corresponding peptide variants in the PREFER plasma proteome samples. The y -axis shows the frequency in percentage of total, and the x -axis shows the corresponding genomic allele and peptide variant combinations. A comparison of the frequencies for each allele was made using Fisher’s Exact Test, which is shown at the top of the plot.
    Proteomics Grade Trypsin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 502 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore trypsin sepharose 4b
    Purification profile of trypsin-specific PIs (CpPI 63), Gelatin native PAGE, and Tricine SDS-PAGE . Elution profile of (A) DEAE-cellulose column loaded with 20–70% (NH 4 ) 2 SO 4 fraction; (B) <t>trypsin-Sepharose</t> 4B column loaded with PI fraction (No. 20–63) pool from the ion-exchange column; (C) Sephadex G-50 column loaded with PI fraction (No. 12–18) pool from the affinity column; (D) activity staining of Gelatin native PAGE (12.5%) and; (E) Tricine SDS-PAGE (18%) showing different fractions of the purification protocol. Lanes 1-6 are loaded with crude PI protein extract, 20–70% (NH 4 ) 2 SO 4 protein fraction, active fraction pool against trypsin from anion exchange column, affinity column, gel-filtration column (fraction No. 38–73), Marker protein, soybean BBI (2 μg) or Puregene molecular weight marker, respectively. Gels were stained with CBB R250 stain or silver nitrate; (F) Tricine SDS-PAGE (18%) showing the consequent increase in intensity of PI bands and appearance of their oligomers with the progressive increase in concentration of CpPI 63 (lanes 3–7). Lanes 1 and 2 are loaded with molecular weight marker (Puregene) and soybean BBI (2 μg), a PI marker, as the mobility of PIs is known to vary as compared to other proteins. The molecular mass of monomeric (8 kDa), dimeric (16 kDa), and trimeric (24 kDa) forms of BBI is indicated. An additional band at ~19 kDa might be a contamination of Kunitz inhibitor in BBI preparation, which is procured commercially. The data shown here are representative of a purification protocol for CpPI 63 from four experiments performed independently as described in Table 1 .
    Trypsin Sepharose 4b, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher trypsin
    Purification profile of trypsin-specific PIs (CpPI 63), Gelatin native PAGE, and Tricine SDS-PAGE . Elution profile of (A) DEAE-cellulose column loaded with 20–70% (NH 4 ) 2 SO 4 fraction; (B) <t>trypsin-Sepharose</t> 4B column loaded with PI fraction (No. 20–63) pool from the ion-exchange column; (C) Sephadex G-50 column loaded with PI fraction (No. 12–18) pool from the affinity column; (D) activity staining of Gelatin native PAGE (12.5%) and; (E) Tricine SDS-PAGE (18%) showing different fractions of the purification protocol. Lanes 1-6 are loaded with crude PI protein extract, 20–70% (NH 4 ) 2 SO 4 protein fraction, active fraction pool against trypsin from anion exchange column, affinity column, gel-filtration column (fraction No. 38–73), Marker protein, soybean BBI (2 μg) or Puregene molecular weight marker, respectively. Gels were stained with CBB R250 stain or silver nitrate; (F) Tricine SDS-PAGE (18%) showing the consequent increase in intensity of PI bands and appearance of their oligomers with the progressive increase in concentration of CpPI 63 (lanes 3–7). Lanes 1 and 2 are loaded with molecular weight marker (Puregene) and soybean BBI (2 μg), a PI marker, as the mobility of PIs is known to vary as compared to other proteins. The molecular mass of monomeric (8 kDa), dimeric (16 kDa), and trimeric (24 kDa) forms of BBI is indicated. An additional band at ~19 kDa might be a contamination of Kunitz inhibitor in BBI preparation, which is procured commercially. The data shown here are representative of a purification protocol for CpPI 63 from four experiments performed independently as described in Table 1 .
    Trypsin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 23190 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher trypsin inhibitor
    Purification profile of trypsin-specific PIs (CpPI 63), Gelatin native PAGE, and Tricine SDS-PAGE . Elution profile of (A) DEAE-cellulose column loaded with 20–70% (NH 4 ) 2 SO 4 fraction; (B) <t>trypsin-Sepharose</t> 4B column loaded with PI fraction (No. 20–63) pool from the ion-exchange column; (C) Sephadex G-50 column loaded with PI fraction (No. 12–18) pool from the affinity column; (D) activity staining of Gelatin native PAGE (12.5%) and; (E) Tricine SDS-PAGE (18%) showing different fractions of the purification protocol. Lanes 1-6 are loaded with crude PI protein extract, 20–70% (NH 4 ) 2 SO 4 protein fraction, active fraction pool against trypsin from anion exchange column, affinity column, gel-filtration column (fraction No. 38–73), Marker protein, soybean BBI (2 μg) or Puregene molecular weight marker, respectively. Gels were stained with CBB R250 stain or silver nitrate; (F) Tricine SDS-PAGE (18%) showing the consequent increase in intensity of PI bands and appearance of their oligomers with the progressive increase in concentration of CpPI 63 (lanes 3–7). Lanes 1 and 2 are loaded with molecular weight marker (Puregene) and soybean BBI (2 μg), a PI marker, as the mobility of PIs is known to vary as compared to other proteins. The molecular mass of monomeric (8 kDa), dimeric (16 kDa), and trimeric (24 kDa) forms of BBI is indicated. An additional band at ~19 kDa might be a contamination of Kunitz inhibitor in BBI preparation, which is procured commercially. The data shown here are representative of a purification protocol for CpPI 63 from four experiments performed independently as described in Table 1 .
    Trypsin Inhibitor, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 910 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Worthington Biochemical trypsin inhibitor
    Purification profile of trypsin-specific PIs (CpPI 63), Gelatin native PAGE, and Tricine SDS-PAGE . Elution profile of (A) DEAE-cellulose column loaded with 20–70% (NH 4 ) 2 SO 4 fraction; (B) <t>trypsin-Sepharose</t> 4B column loaded with PI fraction (No. 20–63) pool from the ion-exchange column; (C) Sephadex G-50 column loaded with PI fraction (No. 12–18) pool from the affinity column; (D) activity staining of Gelatin native PAGE (12.5%) and; (E) Tricine SDS-PAGE (18%) showing different fractions of the purification protocol. Lanes 1-6 are loaded with crude PI protein extract, 20–70% (NH 4 ) 2 SO 4 protein fraction, active fraction pool against trypsin from anion exchange column, affinity column, gel-filtration column (fraction No. 38–73), Marker protein, soybean BBI (2 μg) or Puregene molecular weight marker, respectively. Gels were stained with CBB R250 stain or silver nitrate; (F) Tricine SDS-PAGE (18%) showing the consequent increase in intensity of PI bands and appearance of their oligomers with the progressive increase in concentration of CpPI 63 (lanes 3–7). Lanes 1 and 2 are loaded with molecular weight marker (Puregene) and soybean BBI (2 μg), a PI marker, as the mobility of PIs is known to vary as compared to other proteins. The molecular mass of monomeric (8 kDa), dimeric (16 kDa), and trimeric (24 kDa) forms of BBI is indicated. An additional band at ~19 kDa might be a contamination of Kunitz inhibitor in BBI preparation, which is procured commercially. The data shown here are representative of a purification protocol for CpPI 63 from four experiments performed independently as described in Table 1 .
    Trypsin Inhibitor, supplied by Worthington Biochemical, used in various techniques. Bioz Stars score: 99/100, based on 150 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Inhibition of KSHV infection by anti-integrin antibodies and soluble integrins. (A) Inhibition of GFP-KSHV infection by anti-integrin antibodies. Monolayers of HMVEC-d and HFF were incubated with 10 μg/ml of MAbs against integrins or integrin subunits for 1 h at 4°C, washed, and infected with GFP-KSHV at an MOI of 5 DNA copies/cell. After incubation for 2 h at 37°C, the cells were washed and incubated for 72 h, and the number of GFP-positive cells was estimated under a fluorescence microscope. Each experiment was done in duplicate, and each bar represents the average plus the standard deviation (SD) of three experiments. (B) Effects of anti-integrin antibodies on KSHV binding. HFF were incubated with anti-integrin antibodies (10 μg/ml) at 4°C for 90 min and incubated with a predetermined concentration of [ 3 H]thymidine-labeled purified KSHV. The labeled KSHV was also mixed with 10 μg/ml of heparin or soluble integrins for 90 min at 4°C and then added to the cells. After incubation for 90 min at 4°C with the virus, the cells were washed, lysed, precipitated with trichloroacetic acid, and counted. The bound virus cpm in the absence of any treatment was considered to be 100% and was used to calculate the percentage inhibition of virus binding. Each reaction was done in triplicate, and each bar represents the average plus the SD of three independent experiments. The results with HFF are shown. (C) Inhibition of KSHV infection by soluble integrins. KSHV (5 DNA copies/cell) was mixed with 10 μg/ml of heparin or 10 μg/ml soluble integrins for 2 h at 37°C and added to the cells. After 2 h, the cells were washed to remove the unbound virus, treated with 0.25% trypsin-EDTA for 5 min at 37°C to remove the bound but noninternalized viruses, and washed, and total DNA was isolated. The number of KSHV ORF73 copies was estimated by real-time DNA PCR. The cycle threshold values were used to plot the standard graph and to calculate the relative copy numbers of viral DNA in the samples. The data are presented as percentages of inhibition of KSHV DNA internalization obtained when the cells were incubated with the virus alone. Each reaction was done in duplicate, and each bar represents the average ± SD of three experiments. (D) KSHV was mixed with 10 μg/ml soluble integrin combinations for 2 h at 37°C and added to HFF. After 2 h, the cells were washed to remove the unbound virus and treated with 0.25% trypsin-EDTA for 5 min at 37°C to remove the bound but noninternalized viruses, and the internalized viral DNA was estimated by real-time DNA PCR. Each reaction was done in duplicate, and each bar represents the average ± the SD of three experiments.

    Journal: Journal of Virology

    Article Title: Kaposi's Sarcoma-Associated Herpesvirus Forms a Multimolecular Complex of Integrins (?V?5, ?V?3, and ?3?1) and CD98-xCT during Infection of Human Dermal Microvascular Endothelial Cells, and CD98-xCT Is Essential for the Postentry Stage of Infection ▿

    doi: 10.1128/JVI.01146-08

    Figure Lengend Snippet: Inhibition of KSHV infection by anti-integrin antibodies and soluble integrins. (A) Inhibition of GFP-KSHV infection by anti-integrin antibodies. Monolayers of HMVEC-d and HFF were incubated with 10 μg/ml of MAbs against integrins or integrin subunits for 1 h at 4°C, washed, and infected with GFP-KSHV at an MOI of 5 DNA copies/cell. After incubation for 2 h at 37°C, the cells were washed and incubated for 72 h, and the number of GFP-positive cells was estimated under a fluorescence microscope. Each experiment was done in duplicate, and each bar represents the average plus the standard deviation (SD) of three experiments. (B) Effects of anti-integrin antibodies on KSHV binding. HFF were incubated with anti-integrin antibodies (10 μg/ml) at 4°C for 90 min and incubated with a predetermined concentration of [ 3 H]thymidine-labeled purified KSHV. The labeled KSHV was also mixed with 10 μg/ml of heparin or soluble integrins for 90 min at 4°C and then added to the cells. After incubation for 90 min at 4°C with the virus, the cells were washed, lysed, precipitated with trichloroacetic acid, and counted. The bound virus cpm in the absence of any treatment was considered to be 100% and was used to calculate the percentage inhibition of virus binding. Each reaction was done in triplicate, and each bar represents the average plus the SD of three independent experiments. The results with HFF are shown. (C) Inhibition of KSHV infection by soluble integrins. KSHV (5 DNA copies/cell) was mixed with 10 μg/ml of heparin or 10 μg/ml soluble integrins for 2 h at 37°C and added to the cells. After 2 h, the cells were washed to remove the unbound virus, treated with 0.25% trypsin-EDTA for 5 min at 37°C to remove the bound but noninternalized viruses, and washed, and total DNA was isolated. The number of KSHV ORF73 copies was estimated by real-time DNA PCR. The cycle threshold values were used to plot the standard graph and to calculate the relative copy numbers of viral DNA in the samples. The data are presented as percentages of inhibition of KSHV DNA internalization obtained when the cells were incubated with the virus alone. Each reaction was done in duplicate, and each bar represents the average ± SD of three experiments. (D) KSHV was mixed with 10 μg/ml soluble integrin combinations for 2 h at 37°C and added to HFF. After 2 h, the cells were washed to remove the unbound virus and treated with 0.25% trypsin-EDTA for 5 min at 37°C to remove the bound but noninternalized viruses, and the internalized viral DNA was estimated by real-time DNA PCR. Each reaction was done in duplicate, and each bar represents the average ± the SD of three experiments.

    Article Snippet: Briefly, cells infected with KSHV were collected after 2 h, washed, treated with trypsin-EDTA (0.25% trypsin and 5 mM EDTA) to remove noninternalized virus, and lysed on ice for 5 min with a mild lysis buffer (Sigma), and the nuclei were concentrated by centrifugation at 500 × g for 5 min. DNA was isolated from the nuclei using a DNeasy kit (Qiagen) as described previously ( ).

    Techniques: Inhibition, Infection, Incubation, Fluorescence, Microscopy, Standard Deviation, Binding Assay, Concentration Assay, Labeling, Purification, Isolation, Polymerase Chain Reaction

    ( A) Effects of anti-CD98 and anti-xCT peptide antibodies on KSHV binding. HMVEC-d, untreated or treated with 10 μg/ml of various antibodies at 4°C for 1 h, were incubated with a fixed concentration of [ 3 H]thymidine-labeled virus in RPMI 1640 for 1 h at 4°C with gentle rotation. For control, [ 3 H]thymidine-labeled KSHV was preincubated with 100 μg of heparin/ml for 1 h at 37°C before addition to the cells. After incubation at 4°C, the cells were washed, lysed, and precipitated with trichloroacetic acid, and cell-associated virus radioactivity (in cpm) was measured. The cpm of bound virus in the absence of any treatment was considered to be 100% and was used to calculate the percentage inhibition of virus binding. Each reaction was done in triplicate, and each bar represents the average ± standard deviation (SD) of three independent experiments. (B and C) Effects of anti-CD98 and anti-xCT peptide antibodies on KSHV DNA internalization. HMVEC-d were untreated or treated with various concentrations of anti-CD98 MAb (CD98 MAb) and polyclonal antibody (CD98 PAb) or anti-CD71 antibodies (B) or with anti-xCT peptide antibodies (C) at 4°C for 1 h and then infected with KSHV at an MOI of 10 for 2 h. Unbound virus was removed by washing the cells, and the cells were treated with 0.25% trypsin-EDTA for 5 min at 37°C to remove the bound but noninternalized virus. The cells were washed, DNA was extracted, and the internalized KSHV DNA was quantitated by amplification of the ORF73 gene by real-time DNA PCR. Copy numbers were calculated from the standard graph generated by real-time DNA PCR using known concentrations of a cloned ORF73 gene. Each reaction was done in duplicate, and each bar represents the average ± SD of three experiments. (D) Effect of CD98 ligand, galectin 3, on KSHV DNA internalization. HMVEC-d were first incubated with the CD98 ligand galectin at 5- and 10-μg/ml concentrations for 1 h at 4°C. The cells were washed and then infected with KSHV at an MOI of 10 for 2 h. Internalized KSHV DNA was quantitated by amplification of the ORF73 gene by real-time DNA PCR. Each reaction was done in duplicate, and each bar represents the average ± the SD of three experiments. KSHV DNA internalization in the absence of any treatment was considered to be 100%.

    Journal: Journal of Virology

    Article Title: Kaposi's Sarcoma-Associated Herpesvirus Forms a Multimolecular Complex of Integrins (?V?5, ?V?3, and ?3?1) and CD98-xCT during Infection of Human Dermal Microvascular Endothelial Cells, and CD98-xCT Is Essential for the Postentry Stage of Infection ▿

    doi: 10.1128/JVI.01146-08

    Figure Lengend Snippet: ( A) Effects of anti-CD98 and anti-xCT peptide antibodies on KSHV binding. HMVEC-d, untreated or treated with 10 μg/ml of various antibodies at 4°C for 1 h, were incubated with a fixed concentration of [ 3 H]thymidine-labeled virus in RPMI 1640 for 1 h at 4°C with gentle rotation. For control, [ 3 H]thymidine-labeled KSHV was preincubated with 100 μg of heparin/ml for 1 h at 37°C before addition to the cells. After incubation at 4°C, the cells were washed, lysed, and precipitated with trichloroacetic acid, and cell-associated virus radioactivity (in cpm) was measured. The cpm of bound virus in the absence of any treatment was considered to be 100% and was used to calculate the percentage inhibition of virus binding. Each reaction was done in triplicate, and each bar represents the average ± standard deviation (SD) of three independent experiments. (B and C) Effects of anti-CD98 and anti-xCT peptide antibodies on KSHV DNA internalization. HMVEC-d were untreated or treated with various concentrations of anti-CD98 MAb (CD98 MAb) and polyclonal antibody (CD98 PAb) or anti-CD71 antibodies (B) or with anti-xCT peptide antibodies (C) at 4°C for 1 h and then infected with KSHV at an MOI of 10 for 2 h. Unbound virus was removed by washing the cells, and the cells were treated with 0.25% trypsin-EDTA for 5 min at 37°C to remove the bound but noninternalized virus. The cells were washed, DNA was extracted, and the internalized KSHV DNA was quantitated by amplification of the ORF73 gene by real-time DNA PCR. Copy numbers were calculated from the standard graph generated by real-time DNA PCR using known concentrations of a cloned ORF73 gene. Each reaction was done in duplicate, and each bar represents the average ± SD of three experiments. (D) Effect of CD98 ligand, galectin 3, on KSHV DNA internalization. HMVEC-d were first incubated with the CD98 ligand galectin at 5- and 10-μg/ml concentrations for 1 h at 4°C. The cells were washed and then infected with KSHV at an MOI of 10 for 2 h. Internalized KSHV DNA was quantitated by amplification of the ORF73 gene by real-time DNA PCR. Each reaction was done in duplicate, and each bar represents the average ± the SD of three experiments. KSHV DNA internalization in the absence of any treatment was considered to be 100%.

    Article Snippet: Briefly, cells infected with KSHV were collected after 2 h, washed, treated with trypsin-EDTA (0.25% trypsin and 5 mM EDTA) to remove noninternalized virus, and lysed on ice for 5 min with a mild lysis buffer (Sigma), and the nuclei were concentrated by centrifugation at 500 × g for 5 min. DNA was isolated from the nuclei using a DNeasy kit (Qiagen) as described previously ( ).

    Techniques: Binding Assay, Incubation, Concentration Assay, Labeling, Radioactivity, Inhibition, Standard Deviation, Infection, Amplification, Polymerase Chain Reaction, Generated, Clone Assay

    The HPLC elution patterns of Kunitz trypsin inhibitor (KTI), lectin and soybean whey.

    Journal: International Journal of Molecular Sciences

    Article Title: Selective Isolation of Trypsin Inhibitor and Lectin from Soybean Whey by Chitosan/Tripolyphosphate/Genipin Co-Crosslinked Beads

    doi: 10.3390/ijms15069979

    Figure Lengend Snippet: The HPLC elution patterns of Kunitz trypsin inhibitor (KTI), lectin and soybean whey.

    Article Snippet: Sodium tripolyphosphate (TPP), sodium hydroxide, sodium azide, Kunitz trypsin inhibitor, lectin, and sodium acetate anhydrous were purchased from Sigma-Aldrich (St. Louis, MO, USA).

    Techniques: High Performance Liquid Chromatography

    ISP enzyme kinetics . A- Affinity of different synthetic chromogenic substrates (N-Benzoyl-DL-Arginine-β-Naphthylamide [BANA], N-Benzoyl-DL-Arginine-p-NA [BAPNA], N-Benzoyl-L-Arginine Ethyl Ester [BAEE], N-Benzoyl-Phe-Val-Arg-pNA [BPVANA], N-Benzoyl-L-Tyrosine-pNA [BTNA], N-Succinyl-L-Phe-pNA [SPNA], N-Succinyl-Ala-Ala-Ala-pNA [SAANA], N-CBZ-Gly-Gly-Leu-pNA [CGGLNA], Ile-Pro-Lys-pNA [IPLNA], N-Tosyl-Gly-Pro-Lys-pNA [TGPLNA], Val-Pro-Lys-pNA [VPLNA]) for the ISP1-ISP2 enzyme complex. The trypsin substrate BPVANA showed the greatest affinity to the enzyme complex. B- Enzyme inhibition data showing the affinity of different serine proteinase inhibitors to the ISP1-ISP2 enzyme complex. TSI, Gabexate and BABIM have a relatively high affinity for ISP enzyme, compared to Benzamidine.

    Journal: BMC Developmental Biology

    Article Title: Implantation Serine Proteinases heterodimerize and are critical in hatching and implantation

    doi: 10.1186/1471-213X-6-61

    Figure Lengend Snippet: ISP enzyme kinetics . A- Affinity of different synthetic chromogenic substrates (N-Benzoyl-DL-Arginine-β-Naphthylamide [BANA], N-Benzoyl-DL-Arginine-p-NA [BAPNA], N-Benzoyl-L-Arginine Ethyl Ester [BAEE], N-Benzoyl-Phe-Val-Arg-pNA [BPVANA], N-Benzoyl-L-Tyrosine-pNA [BTNA], N-Succinyl-L-Phe-pNA [SPNA], N-Succinyl-Ala-Ala-Ala-pNA [SAANA], N-CBZ-Gly-Gly-Leu-pNA [CGGLNA], Ile-Pro-Lys-pNA [IPLNA], N-Tosyl-Gly-Pro-Lys-pNA [TGPLNA], Val-Pro-Lys-pNA [VPLNA]) for the ISP1-ISP2 enzyme complex. The trypsin substrate BPVANA showed the greatest affinity to the enzyme complex. B- Enzyme inhibition data showing the affinity of different serine proteinase inhibitors to the ISP1-ISP2 enzyme complex. TSI, Gabexate and BABIM have a relatively high affinity for ISP enzyme, compared to Benzamidine.

    Article Snippet: Substrates and Inhibitors p-Nitroanilide substrates, Benzamidine Hydrochloride, TLCK (N -p-tosyl-L-lysine chloromethyl ketone), APMSF (4-Amidino-Phenyl)-Methane-Sulfonyl Fluoride), Trypsin Soybean Inhibitor (TSI) and Gabexate mesylate were obtained from Sigma Aldrich and Co. Dr. George H. Caughey, University of California, San Francisco kindly supplied BABIM.

    Techniques: Enzyme Inhibition Assay

    Detection of clinically relevant natural protein variants in the PREFER plasma proteomes. Mass spectrometry-based proteomics data was reanalyzed using a modified plasma-specific protein database containing all known forms of naturally occurring clinically-relevant variants. (a) Bar plot for each protein variant where the y -axis shows the summed intensity of peptides derived from each allele as a percentage of total, and the x -axis shows the 44 participant samples with one bar per sample. The gene name for each variant and its position in the protein are indicated in bold to the right of each plot. Also shown are the associated human phenotypes. Peptides from each allele are shown in different colors on the same plot and correspond to each legend shown on the right. (b) Bar plot showing either the frequency of the genotype in the European population sampled by the 1000Genomes study, or the frequency of the corresponding peptide variants in the PREFER plasma proteome samples. The y -axis shows the frequency in percentage of total, and the x -axis shows the corresponding genomic allele and peptide variant combinations. A comparison of the frequencies for each allele was made using Fisher’s Exact Test, which is shown at the top of the plot.

    Journal: Journal of Proteome Research

    Article Title: Proteomic Analysis of Human Plasma during Intermittent Fasting

    doi: 10.1021/acs.jproteome.9b00090

    Figure Lengend Snippet: Detection of clinically relevant natural protein variants in the PREFER plasma proteomes. Mass spectrometry-based proteomics data was reanalyzed using a modified plasma-specific protein database containing all known forms of naturally occurring clinically-relevant variants. (a) Bar plot for each protein variant where the y -axis shows the summed intensity of peptides derived from each allele as a percentage of total, and the x -axis shows the 44 participant samples with one bar per sample. The gene name for each variant and its position in the protein are indicated in bold to the right of each plot. Also shown are the associated human phenotypes. Peptides from each allele are shown in different colors on the same plot and correspond to each legend shown on the right. (b) Bar plot showing either the frequency of the genotype in the European population sampled by the 1000Genomes study, or the frequency of the corresponding peptide variants in the PREFER plasma proteome samples. The y -axis shows the frequency in percentage of total, and the x -axis shows the corresponding genomic allele and peptide variant combinations. A comparison of the frequencies for each allele was made using Fisher’s Exact Test, which is shown at the top of the plot.

    Article Snippet: Proteomics-grade trypsin (Catalogue number T6567) and all other reagents were from Sigma-Aldrich (Missouri, USA).

    Techniques: Mass Spectrometry, Modification, Variant Assay, Derivative Assay

    PREFER clinical trial plasma quality control. Plasma from participants before and after IF were analyzed using our Spin96 proteomics workflow. Label-free quantitation (LFQ) intensity was plotted for (a) proteins indicative of blood clotting and hemolysis for each participant, before and after fasting, and (b) LFQ intensity plot for example proteins of very similar abundance before and after the IF intervention, which showed participant-specific abundance patterns. Each color represents a different protein. (c) Plot for each plasma protein of the adjusted LFQ (Adj. LFQ) intensity versus the Adj. LFQ percentage coefficient of variation (% CV). Green circles indicate

    Journal: Journal of Proteome Research

    Article Title: Proteomic Analysis of Human Plasma during Intermittent Fasting

    doi: 10.1021/acs.jproteome.9b00090

    Figure Lengend Snippet: PREFER clinical trial plasma quality control. Plasma from participants before and after IF were analyzed using our Spin96 proteomics workflow. Label-free quantitation (LFQ) intensity was plotted for (a) proteins indicative of blood clotting and hemolysis for each participant, before and after fasting, and (b) LFQ intensity plot for example proteins of very similar abundance before and after the IF intervention, which showed participant-specific abundance patterns. Each color represents a different protein. (c) Plot for each plasma protein of the adjusted LFQ (Adj. LFQ) intensity versus the Adj. LFQ percentage coefficient of variation (% CV). Green circles indicate

    Article Snippet: Proteomics-grade trypsin (Catalogue number T6567) and all other reagents were from Sigma-Aldrich (Missouri, USA).

    Techniques: Quantitation Assay, Coagulation

    Purification profile of trypsin-specific PIs (CpPI 63), Gelatin native PAGE, and Tricine SDS-PAGE . Elution profile of (A) DEAE-cellulose column loaded with 20–70% (NH 4 ) 2 SO 4 fraction; (B) trypsin-Sepharose 4B column loaded with PI fraction (No. 20–63) pool from the ion-exchange column; (C) Sephadex G-50 column loaded with PI fraction (No. 12–18) pool from the affinity column; (D) activity staining of Gelatin native PAGE (12.5%) and; (E) Tricine SDS-PAGE (18%) showing different fractions of the purification protocol. Lanes 1-6 are loaded with crude PI protein extract, 20–70% (NH 4 ) 2 SO 4 protein fraction, active fraction pool against trypsin from anion exchange column, affinity column, gel-filtration column (fraction No. 38–73), Marker protein, soybean BBI (2 μg) or Puregene molecular weight marker, respectively. Gels were stained with CBB R250 stain or silver nitrate; (F) Tricine SDS-PAGE (18%) showing the consequent increase in intensity of PI bands and appearance of their oligomers with the progressive increase in concentration of CpPI 63 (lanes 3–7). Lanes 1 and 2 are loaded with molecular weight marker (Puregene) and soybean BBI (2 μg), a PI marker, as the mobility of PIs is known to vary as compared to other proteins. The molecular mass of monomeric (8 kDa), dimeric (16 kDa), and trimeric (24 kDa) forms of BBI is indicated. An additional band at ~19 kDa might be a contamination of Kunitz inhibitor in BBI preparation, which is procured commercially. The data shown here are representative of a purification protocol for CpPI 63 from four experiments performed independently as described in Table 1 .

    Journal: Frontiers in Physiology

    Article Title: Purification and Partial Characterization of Trypsin-Specific Proteinase Inhibitors from Pigeonpea Wild Relative Cajanus platycarpus L. (Fabaceae) Active against Gut Proteases of Lepidopteran Pest Helicoverpa armigera

    doi: 10.3389/fphys.2016.00388

    Figure Lengend Snippet: Purification profile of trypsin-specific PIs (CpPI 63), Gelatin native PAGE, and Tricine SDS-PAGE . Elution profile of (A) DEAE-cellulose column loaded with 20–70% (NH 4 ) 2 SO 4 fraction; (B) trypsin-Sepharose 4B column loaded with PI fraction (No. 20–63) pool from the ion-exchange column; (C) Sephadex G-50 column loaded with PI fraction (No. 12–18) pool from the affinity column; (D) activity staining of Gelatin native PAGE (12.5%) and; (E) Tricine SDS-PAGE (18%) showing different fractions of the purification protocol. Lanes 1-6 are loaded with crude PI protein extract, 20–70% (NH 4 ) 2 SO 4 protein fraction, active fraction pool against trypsin from anion exchange column, affinity column, gel-filtration column (fraction No. 38–73), Marker protein, soybean BBI (2 μg) or Puregene molecular weight marker, respectively. Gels were stained with CBB R250 stain or silver nitrate; (F) Tricine SDS-PAGE (18%) showing the consequent increase in intensity of PI bands and appearance of their oligomers with the progressive increase in concentration of CpPI 63 (lanes 3–7). Lanes 1 and 2 are loaded with molecular weight marker (Puregene) and soybean BBI (2 μg), a PI marker, as the mobility of PIs is known to vary as compared to other proteins. The molecular mass of monomeric (8 kDa), dimeric (16 kDa), and trimeric (24 kDa) forms of BBI is indicated. An additional band at ~19 kDa might be a contamination of Kunitz inhibitor in BBI preparation, which is procured commercially. The data shown here are representative of a purification protocol for CpPI 63 from four experiments performed independently as described in Table 1 .

    Article Snippet: DEAE-cellulose, trypsin-Sepharose 4B, Sephadex G-50, N-α-benzoyl-DL-arginine-p -nitroanilide (BAp NA), Bowman-Birk inhibitor from soybean (BBI), sorbitol, tricine, gelatin, and coomassie brilliant blue (CBB) R-250 were purchased from Sigma (St. Louis, MO).

    Techniques: Purification, Clear Native PAGE, SDS Page, Affinity Column, Activity Assay, Staining, Filtration, Marker, Molecular Weight, Concentration Assay