granzyme b substrate acetyl  (Millipore)


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    Name:
    Granzyme B Substrate
    Description:

    Catalog Number:
    scp0158
    Price:
    None
    Applications:
    Granzyme B Substrate (Ac-IEPD-AMC) is a fluorogenic substrate for the detection and assay of caspase 8 and granzyme B which is involved in the rapid induction of target cell apoptosis by CTL in cell-mediated immune response.
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    Structured Review

    Millipore granzyme b substrate acetyl
    (A) Cytotoxic activity and the levels of <t>granzyme</t> B content and release of 12 T. parva -specific CD8 + T cell clones isolated from two animals (animals 641 and 633) were assayed with autologous T. parva -infected target cells. A standard effector-to-target cell ratio of 2:1 was used. (B, C) Correlation of granzyme B cellular activity with the levels of granzyme B released following antigenic stimulation ( r = 0.953, P

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    Price from $9.99 to $1999.99
    granzyme b substrate acetyl - by Bioz Stars, 2020-09
    95/100 stars

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    Images

    1) Product Images from "Granzyme B Is an Essential Mediator in CD8+ T Cell Killing of Theileria parva-Infected Cells"

    Article Title: Granzyme B Is an Essential Mediator in CD8+ T Cell Killing of Theileria parva-Infected Cells

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00386-18

    (A) Cytotoxic activity and the levels of granzyme B content and release of 12 T. parva -specific CD8 + T cell clones isolated from two animals (animals 641 and 633) were assayed with autologous T. parva -infected target cells. A standard effector-to-target cell ratio of 2:1 was used. (B, C) Correlation of granzyme B cellular activity with the levels of granzyme B released following antigenic stimulation ( r = 0.953, P
    Figure Legend Snippet: (A) Cytotoxic activity and the levels of granzyme B content and release of 12 T. parva -specific CD8 + T cell clones isolated from two animals (animals 641 and 633) were assayed with autologous T. parva -infected target cells. A standard effector-to-target cell ratio of 2:1 was used. (B, C) Correlation of granzyme B cellular activity with the levels of granzyme B released following antigenic stimulation ( r = 0.953, P

    Techniques Used: Activity Assay, Clone Assay, Isolation, Infection

    (A) Amino acid sequences from the nucleotide sequences of three recombinant forms of bovine granzyme B cDNA aligned with the reference sequence from the genome database. Granzyme B-CDs, the full-length cDNA from the bovine genome assembly, UMD3.1 (accession number corrected_ENSBTAG00000010057); Granzyme B-WT, the pFLAG-CMV-5a vector containing wild-type granzyme B; Granzyme B-Function, the pFLAG-CMV-5a vector containing functional granzyme B; Granzyme B-Mutant, the pFLAG-CMV-5a vector containing functional granzyme B with a Ser 195 -to-Ala 195 mutation; dots, identical residues; dashes, gaps; red box, leader peptide; yellow box, dipeptide/GE; black box, Ser195Ala; blue box, FLAG epitope tag sequence of the pFLAG-CMV-5a vector. (B) Enzymatic activity of different recombinant forms of bovine granzyme B tested on a granzyme B-specific substrate, Ac-IEPD- p NA (filled bars), and a control substrate, Suc-GGF- p NA (empty bars). Cos-7 cells were transiently transfected with unmodified granzyme B cDNA (WT), cDNA with the GE dipeptide deleted (Function), or cDNA containing a deletion of the dipeptide and an alanine substitution at position 195 (Mutant). The transfection efficiencies of Cos-7 cells with the three granzyme B constructs were 35%, 33%, and 33%, respectively. Lysates of the transfected cells collected after 48 h were incubated with the substrates for 4 h. Controls consisted of lysates of cells transfected with pFLAG without an insert (Mock) and buffer (No cells) added to the substrate. The color reaction generated after 4 h by cleavage of the p NA substrate was measured at a wavelength of 405 nm using a Synergy HT multimode microplate reader (BioTek). (C) Inhibition of the functional recombinant cattle granzyme B by preincubating with 10 μM granzyme B-specific inhibitor Ac-IEPD-CHO for 0.5 h.
    Figure Legend Snippet: (A) Amino acid sequences from the nucleotide sequences of three recombinant forms of bovine granzyme B cDNA aligned with the reference sequence from the genome database. Granzyme B-CDs, the full-length cDNA from the bovine genome assembly, UMD3.1 (accession number corrected_ENSBTAG00000010057); Granzyme B-WT, the pFLAG-CMV-5a vector containing wild-type granzyme B; Granzyme B-Function, the pFLAG-CMV-5a vector containing functional granzyme B; Granzyme B-Mutant, the pFLAG-CMV-5a vector containing functional granzyme B with a Ser 195 -to-Ala 195 mutation; dots, identical residues; dashes, gaps; red box, leader peptide; yellow box, dipeptide/GE; black box, Ser195Ala; blue box, FLAG epitope tag sequence of the pFLAG-CMV-5a vector. (B) Enzymatic activity of different recombinant forms of bovine granzyme B tested on a granzyme B-specific substrate, Ac-IEPD- p NA (filled bars), and a control substrate, Suc-GGF- p NA (empty bars). Cos-7 cells were transiently transfected with unmodified granzyme B cDNA (WT), cDNA with the GE dipeptide deleted (Function), or cDNA containing a deletion of the dipeptide and an alanine substitution at position 195 (Mutant). The transfection efficiencies of Cos-7 cells with the three granzyme B constructs were 35%, 33%, and 33%, respectively. Lysates of the transfected cells collected after 48 h were incubated with the substrates for 4 h. Controls consisted of lysates of cells transfected with pFLAG without an insert (Mock) and buffer (No cells) added to the substrate. The color reaction generated after 4 h by cleavage of the p NA substrate was measured at a wavelength of 405 nm using a Synergy HT multimode microplate reader (BioTek). (C) Inhibition of the functional recombinant cattle granzyme B by preincubating with 10 μM granzyme B-specific inhibitor Ac-IEPD-CHO for 0.5 h.

    Techniques Used: Recombinant, Sequencing, Plasmid Preparation, Functional Assay, Mutagenesis, FLAG-tag, Activity Assay, Transfection, Construct, Incubation, Generated, Inhibition

    Inhibition of the cytotoxic activity of an uncloned (bulk) CD8 + T cell line from animal 011 (A) and three CD8 + T cell lines from animal 592 (B) by incubation with the perforin inhibitor concanamycin A (CMA) and three CD8 + T cell lines from animal 641 by incubation with the granzyme B inhibitor Z-IETD-FMK (C). (A, B) Effectors (1 × 10 4 ) were preincubated with various concentrations of CMA for 2 h and tested in a 4-h cytotoxicity assay with 111 In-labeled autologous TpM target cells and MHC-matched target cells pulsed with a peptide consisting of T. parva antigen Tp2 from residues 49 to 59 (Tp2 49–59 ) (1,000 ng/ml). (C) Three cloned CD8 + T cell lines (1 × 10 4 ) were preincubated for 1 h with 40 μM Z-IETD-FMK and a negative control, Z-VAD-FMK. Labeled target cells alone were also incubated with the inhibitors in the assay. A standard effector-to-target cell ratio of 2:1 was used.
    Figure Legend Snippet: Inhibition of the cytotoxic activity of an uncloned (bulk) CD8 + T cell line from animal 011 (A) and three CD8 + T cell lines from animal 592 (B) by incubation with the perforin inhibitor concanamycin A (CMA) and three CD8 + T cell lines from animal 641 by incubation with the granzyme B inhibitor Z-IETD-FMK (C). (A, B) Effectors (1 × 10 4 ) were preincubated with various concentrations of CMA for 2 h and tested in a 4-h cytotoxicity assay with 111 In-labeled autologous TpM target cells and MHC-matched target cells pulsed with a peptide consisting of T. parva antigen Tp2 from residues 49 to 59 (Tp2 49–59 ) (1,000 ng/ml). (C) Three cloned CD8 + T cell lines (1 × 10 4 ) were preincubated for 1 h with 40 μM Z-IETD-FMK and a negative control, Z-VAD-FMK. Labeled target cells alone were also incubated with the inhibitors in the assay. A standard effector-to-target cell ratio of 2:1 was used.

    Techniques Used: Inhibition, Activity Assay, Incubation, Cytotoxicity Assay, Labeling, Clone Assay, Negative Control

    (A) PCR products obtained for each of the bovine granule enzymes from an uncloned T. parva -specific CD8 + T cell line (from animal 641). The sizes of the PCR products obtained were as follows: granzyme A (lane A), 838 bp; granzyme O (lane O), 849 bp; granzyme B (lane B), 818 bp; granzyme H (lane H), 820 bp; granzyme K (lane K), 889 bp; granzyme M (lane M), 833 bp; perforin (lane PFN), 1,275 bp. Negative controls (primers with no added cDNA template) were included in the lane to the left. (B) Agarose gels showing the PCR products for granzyme B (457 bp), perforin (1,275 bp), and the GAPDH control (304 bp). The numbers of days after antigenic stimulation are shown. (C) Changes in the quantity of the PCR product (vertical axis) at different times following antigenic stimulation, normalized in relation to that of the GAPDH product obtained from the same sample. (D) Cytotoxic activity of 8 T. parva -specific CD8 + T cell clones from two different animals (animals 641 and 011) assayed on autologous T. parva -infected targets. (E) Agarose gels showing the PCR products for granzyme B (457 bp), perforin (1,275 bp), and the GAPDH control (304 bp) from 8 T. parva -specific CD8 + T cell clones (D). (F) Correlation of killing of Theileria- infected target cells by CD8 + T cell clones with the levels of mRNA expression of granzyme B ( r = 0.438, P = 0.278) and perforin ( r = −0.104, P = 0.806). Changes in the quantity of the PCR product (vertical axis) in different T cell clones normalized in relation to that of the GAPDH product obtained from the same sample. (B, E) A negative control (lanes −) without added template and a positive control (lanes +) consisting of primers with a cDNA template of an uncloned T. parva -specific CD8 + T cell line (from animal 641) obtained on day 7 after the 3rd stimulation are included. The density of all PCR amplicon bands was measured by Kodak 1D software (version 3.6). The correlation between variables was analyzed by Pearson’s correlation test. P values of
    Figure Legend Snippet: (A) PCR products obtained for each of the bovine granule enzymes from an uncloned T. parva -specific CD8 + T cell line (from animal 641). The sizes of the PCR products obtained were as follows: granzyme A (lane A), 838 bp; granzyme O (lane O), 849 bp; granzyme B (lane B), 818 bp; granzyme H (lane H), 820 bp; granzyme K (lane K), 889 bp; granzyme M (lane M), 833 bp; perforin (lane PFN), 1,275 bp. Negative controls (primers with no added cDNA template) were included in the lane to the left. (B) Agarose gels showing the PCR products for granzyme B (457 bp), perforin (1,275 bp), and the GAPDH control (304 bp). The numbers of days after antigenic stimulation are shown. (C) Changes in the quantity of the PCR product (vertical axis) at different times following antigenic stimulation, normalized in relation to that of the GAPDH product obtained from the same sample. (D) Cytotoxic activity of 8 T. parva -specific CD8 + T cell clones from two different animals (animals 641 and 011) assayed on autologous T. parva -infected targets. (E) Agarose gels showing the PCR products for granzyme B (457 bp), perforin (1,275 bp), and the GAPDH control (304 bp) from 8 T. parva -specific CD8 + T cell clones (D). (F) Correlation of killing of Theileria- infected target cells by CD8 + T cell clones with the levels of mRNA expression of granzyme B ( r = 0.438, P = 0.278) and perforin ( r = −0.104, P = 0.806). Changes in the quantity of the PCR product (vertical axis) in different T cell clones normalized in relation to that of the GAPDH product obtained from the same sample. (B, E) A negative control (lanes −) without added template and a positive control (lanes +) consisting of primers with a cDNA template of an uncloned T. parva -specific CD8 + T cell line (from animal 641) obtained on day 7 after the 3rd stimulation are included. The density of all PCR amplicon bands was measured by Kodak 1D software (version 3.6). The correlation between variables was analyzed by Pearson’s correlation test. P values of

    Techniques Used: Polymerase Chain Reaction, Activity Assay, Clone Assay, Infection, Expressing, Negative Control, Positive Control, Amplification, Software

    (A) 111 In-labeled peptide-pulsed target cells (5 × 10 3 MHC-matched target cells plus Tp1 214–224 at 100 ng/ml) were preincubated with the pan-caspase inhibitor Z-VAD-FMK (80 μM) for 1 h and tested in a 4-h cytotoxicity assay with two Tp1-specific cloned CD8 + T cell lines from animal 641. As controls, effector cells (1 × 10 4 ) preincubated with the perforin inhibitor CMA (10 ng/ml) for 2 h or the granzyme B inhibitor Z-IETD-FMK (40 μM) for 1 h were tested in the same experiment. Labeled target cells alone were also incubated with these inhibitors in the assay. A standard effector-to-target cell ratio of 2:1 was used. (B) Expression vector pET-15b carrying an N-terminal His tag sequence followed by the full-length coding sequence of bovine Bid was expressed in E. coli BL21(DE3) in the presence (lane +) or absence (lane −) of IPTG, and the expressed products were purified using automated immobilized metal affinity chromatography (IMAC) and automated ion-exchange chromatography (IEC). The products were separated by SDS-PAGE and visualized by Coomassie blue staining. The predicted size of bovine recombinant Bid is 23.7 kDa. (C, D) Purified recombinant bovine Bid proteins (3 μg) were incubated with the indicated concentrations of active bovine granzyme B for 2 h at 37°C. (C) The reaction products were separated by SDS-PAGE and visualized by Coomassie blue staining. (D) Full-length recombinant Bid and truncated Bid (N terminus) were detected by anti-His tag antibody, and recombinant granzyme B was detected by anti-FLAG M2 antibody in a Western blot. Inactive bovine granzyme B mutant cells (with an alanine substitution at position 195), mock-transfected cells (pFLAG without an insert), and Cos-7 cells alone were included as negative controls for granzyme B proteolysis specificity.
    Figure Legend Snippet: (A) 111 In-labeled peptide-pulsed target cells (5 × 10 3 MHC-matched target cells plus Tp1 214–224 at 100 ng/ml) were preincubated with the pan-caspase inhibitor Z-VAD-FMK (80 μM) for 1 h and tested in a 4-h cytotoxicity assay with two Tp1-specific cloned CD8 + T cell lines from animal 641. As controls, effector cells (1 × 10 4 ) preincubated with the perforin inhibitor CMA (10 ng/ml) for 2 h or the granzyme B inhibitor Z-IETD-FMK (40 μM) for 1 h were tested in the same experiment. Labeled target cells alone were also incubated with these inhibitors in the assay. A standard effector-to-target cell ratio of 2:1 was used. (B) Expression vector pET-15b carrying an N-terminal His tag sequence followed by the full-length coding sequence of bovine Bid was expressed in E. coli BL21(DE3) in the presence (lane +) or absence (lane −) of IPTG, and the expressed products were purified using automated immobilized metal affinity chromatography (IMAC) and automated ion-exchange chromatography (IEC). The products were separated by SDS-PAGE and visualized by Coomassie blue staining. The predicted size of bovine recombinant Bid is 23.7 kDa. (C, D) Purified recombinant bovine Bid proteins (3 μg) were incubated with the indicated concentrations of active bovine granzyme B for 2 h at 37°C. (C) The reaction products were separated by SDS-PAGE and visualized by Coomassie blue staining. (D) Full-length recombinant Bid and truncated Bid (N terminus) were detected by anti-His tag antibody, and recombinant granzyme B was detected by anti-FLAG M2 antibody in a Western blot. Inactive bovine granzyme B mutant cells (with an alanine substitution at position 195), mock-transfected cells (pFLAG without an insert), and Cos-7 cells alone were included as negative controls for granzyme B proteolysis specificity.

    Techniques Used: Labeling, Cytotoxicity Assay, Clone Assay, Incubation, Expressing, Plasmid Preparation, Positron Emission Tomography, Sequencing, Purification, Affinity Chromatography, Ion Exchange Chromatography, SDS Page, Staining, Recombinant, Western Blot, Mutagenesis, Transfection

    2) Product Images from "Granzyme B Is an Essential Mediator in CD8+ T Cell Killing of Theileria parva-Infected Cells"

    Article Title: Granzyme B Is an Essential Mediator in CD8+ T Cell Killing of Theileria parva-Infected Cells

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00386-18

    (A) Cytotoxic activity and the levels of granzyme B content and release of 12 T. parva -specific CD8 + T cell clones isolated from two animals (animals 641 and 633) were assayed with autologous T. parva -infected target cells. A standard effector-to-target cell ratio of 2:1 was used. (B, C) Correlation of granzyme B cellular activity with the levels of granzyme B released following antigenic stimulation ( r = 0.953, P
    Figure Legend Snippet: (A) Cytotoxic activity and the levels of granzyme B content and release of 12 T. parva -specific CD8 + T cell clones isolated from two animals (animals 641 and 633) were assayed with autologous T. parva -infected target cells. A standard effector-to-target cell ratio of 2:1 was used. (B, C) Correlation of granzyme B cellular activity with the levels of granzyme B released following antigenic stimulation ( r = 0.953, P

    Techniques Used: Activity Assay, Clone Assay, Isolation, Infection

    (A) Amino acid sequences from the nucleotide sequences of three recombinant forms of bovine granzyme B cDNA aligned with the reference sequence from the genome database. Granzyme B-CDs, the full-length cDNA from the bovine genome assembly, UMD3.1 (accession number corrected_ENSBTAG00000010057); Granzyme B-WT, the pFLAG-CMV-5a vector containing wild-type granzyme B; Granzyme B-Function, the pFLAG-CMV-5a vector containing functional granzyme B; Granzyme B-Mutant, the pFLAG-CMV-5a vector containing functional granzyme B with a Ser 195 -to-Ala 195 mutation; dots, identical residues; dashes, gaps; red box, leader peptide; yellow box, dipeptide/GE; black box, Ser195Ala; blue box, FLAG epitope tag sequence of the pFLAG-CMV-5a vector. (B) Enzymatic activity of different recombinant forms of bovine granzyme B tested on a granzyme B-specific substrate, Ac-IEPD- p NA (filled bars), and a control substrate, Suc-GGF- p NA (empty bars). Cos-7 cells were transiently transfected with unmodified granzyme B cDNA (WT), cDNA with the GE dipeptide deleted (Function), or cDNA containing a deletion of the dipeptide and an alanine substitution at position 195 (Mutant). The transfection efficiencies of Cos-7 cells with the three granzyme B constructs were 35%, 33%, and 33%, respectively. Lysates of the transfected cells collected after 48 h were incubated with the substrates for 4 h. Controls consisted of lysates of cells transfected with pFLAG without an insert (Mock) and buffer (No cells) added to the substrate. The color reaction generated after 4 h by cleavage of the p NA substrate was measured at a wavelength of 405 nm using a Synergy HT multimode microplate reader (BioTek). (C) Inhibition of the functional recombinant cattle granzyme B by preincubating with 10 μM granzyme B-specific inhibitor Ac-IEPD-CHO for 0.5 h.
    Figure Legend Snippet: (A) Amino acid sequences from the nucleotide sequences of three recombinant forms of bovine granzyme B cDNA aligned with the reference sequence from the genome database. Granzyme B-CDs, the full-length cDNA from the bovine genome assembly, UMD3.1 (accession number corrected_ENSBTAG00000010057); Granzyme B-WT, the pFLAG-CMV-5a vector containing wild-type granzyme B; Granzyme B-Function, the pFLAG-CMV-5a vector containing functional granzyme B; Granzyme B-Mutant, the pFLAG-CMV-5a vector containing functional granzyme B with a Ser 195 -to-Ala 195 mutation; dots, identical residues; dashes, gaps; red box, leader peptide; yellow box, dipeptide/GE; black box, Ser195Ala; blue box, FLAG epitope tag sequence of the pFLAG-CMV-5a vector. (B) Enzymatic activity of different recombinant forms of bovine granzyme B tested on a granzyme B-specific substrate, Ac-IEPD- p NA (filled bars), and a control substrate, Suc-GGF- p NA (empty bars). Cos-7 cells were transiently transfected with unmodified granzyme B cDNA (WT), cDNA with the GE dipeptide deleted (Function), or cDNA containing a deletion of the dipeptide and an alanine substitution at position 195 (Mutant). The transfection efficiencies of Cos-7 cells with the three granzyme B constructs were 35%, 33%, and 33%, respectively. Lysates of the transfected cells collected after 48 h were incubated with the substrates for 4 h. Controls consisted of lysates of cells transfected with pFLAG without an insert (Mock) and buffer (No cells) added to the substrate. The color reaction generated after 4 h by cleavage of the p NA substrate was measured at a wavelength of 405 nm using a Synergy HT multimode microplate reader (BioTek). (C) Inhibition of the functional recombinant cattle granzyme B by preincubating with 10 μM granzyme B-specific inhibitor Ac-IEPD-CHO for 0.5 h.

    Techniques Used: Recombinant, Sequencing, Plasmid Preparation, Functional Assay, Mutagenesis, FLAG-tag, Activity Assay, Transfection, Construct, Incubation, Generated, Inhibition

    Inhibition of the cytotoxic activity of an uncloned (bulk) CD8 + T cell line from animal 011 (A) and three CD8 + T cell lines from animal 592 (B) by incubation with the perforin inhibitor concanamycin A (CMA) and three CD8 + T cell lines from animal 641 by incubation with the granzyme B inhibitor Z-IETD-FMK (C). (A, B) Effectors (1 × 10 4 ) were preincubated with various concentrations of CMA for 2 h and tested in a 4-h cytotoxicity assay with 111 In-labeled autologous TpM target cells and MHC-matched target cells pulsed with a peptide consisting of T. parva antigen Tp2 from residues 49 to 59 (Tp2 49–59 ) (1,000 ng/ml). (C) Three cloned CD8 + T cell lines (1 × 10 4 ) were preincubated for 1 h with 40 μM Z-IETD-FMK and a negative control, Z-VAD-FMK. Labeled target cells alone were also incubated with the inhibitors in the assay. A standard effector-to-target cell ratio of 2:1 was used.
    Figure Legend Snippet: Inhibition of the cytotoxic activity of an uncloned (bulk) CD8 + T cell line from animal 011 (A) and three CD8 + T cell lines from animal 592 (B) by incubation with the perforin inhibitor concanamycin A (CMA) and three CD8 + T cell lines from animal 641 by incubation with the granzyme B inhibitor Z-IETD-FMK (C). (A, B) Effectors (1 × 10 4 ) were preincubated with various concentrations of CMA for 2 h and tested in a 4-h cytotoxicity assay with 111 In-labeled autologous TpM target cells and MHC-matched target cells pulsed with a peptide consisting of T. parva antigen Tp2 from residues 49 to 59 (Tp2 49–59 ) (1,000 ng/ml). (C) Three cloned CD8 + T cell lines (1 × 10 4 ) were preincubated for 1 h with 40 μM Z-IETD-FMK and a negative control, Z-VAD-FMK. Labeled target cells alone were also incubated with the inhibitors in the assay. A standard effector-to-target cell ratio of 2:1 was used.

    Techniques Used: Inhibition, Activity Assay, Incubation, Cytotoxicity Assay, Labeling, Clone Assay, Negative Control

    (A) PCR products obtained for each of the bovine granule enzymes from an uncloned T. parva -specific CD8 + T cell line (from animal 641). The sizes of the PCR products obtained were as follows: granzyme A (lane A), 838 bp; granzyme O (lane O), 849 bp; granzyme B (lane B), 818 bp; granzyme H (lane H), 820 bp; granzyme K (lane K), 889 bp; granzyme M (lane M), 833 bp; perforin (lane PFN), 1,275 bp. Negative controls (primers with no added cDNA template) were included in the lane to the left. (B) Agarose gels showing the PCR products for granzyme B (457 bp), perforin (1,275 bp), and the GAPDH control (304 bp). The numbers of days after antigenic stimulation are shown. (C) Changes in the quantity of the PCR product (vertical axis) at different times following antigenic stimulation, normalized in relation to that of the GAPDH product obtained from the same sample. (D) Cytotoxic activity of 8 T. parva -specific CD8 + T cell clones from two different animals (animals 641 and 011) assayed on autologous T. parva -infected targets. (E) Agarose gels showing the PCR products for granzyme B (457 bp), perforin (1,275 bp), and the GAPDH control (304 bp) from 8 T. parva -specific CD8 + T cell clones (D). (F) Correlation of killing of Theileria- infected target cells by CD8 + T cell clones with the levels of mRNA expression of granzyme B ( r = 0.438, P = 0.278) and perforin ( r = −0.104, P = 0.806). Changes in the quantity of the PCR product (vertical axis) in different T cell clones normalized in relation to that of the GAPDH product obtained from the same sample. (B, E) A negative control (lanes −) without added template and a positive control (lanes +) consisting of primers with a cDNA template of an uncloned T. parva -specific CD8 + T cell line (from animal 641) obtained on day 7 after the 3rd stimulation are included. The density of all PCR amplicon bands was measured by Kodak 1D software (version 3.6). The correlation between variables was analyzed by Pearson’s correlation test. P values of
    Figure Legend Snippet: (A) PCR products obtained for each of the bovine granule enzymes from an uncloned T. parva -specific CD8 + T cell line (from animal 641). The sizes of the PCR products obtained were as follows: granzyme A (lane A), 838 bp; granzyme O (lane O), 849 bp; granzyme B (lane B), 818 bp; granzyme H (lane H), 820 bp; granzyme K (lane K), 889 bp; granzyme M (lane M), 833 bp; perforin (lane PFN), 1,275 bp. Negative controls (primers with no added cDNA template) were included in the lane to the left. (B) Agarose gels showing the PCR products for granzyme B (457 bp), perforin (1,275 bp), and the GAPDH control (304 bp). The numbers of days after antigenic stimulation are shown. (C) Changes in the quantity of the PCR product (vertical axis) at different times following antigenic stimulation, normalized in relation to that of the GAPDH product obtained from the same sample. (D) Cytotoxic activity of 8 T. parva -specific CD8 + T cell clones from two different animals (animals 641 and 011) assayed on autologous T. parva -infected targets. (E) Agarose gels showing the PCR products for granzyme B (457 bp), perforin (1,275 bp), and the GAPDH control (304 bp) from 8 T. parva -specific CD8 + T cell clones (D). (F) Correlation of killing of Theileria- infected target cells by CD8 + T cell clones with the levels of mRNA expression of granzyme B ( r = 0.438, P = 0.278) and perforin ( r = −0.104, P = 0.806). Changes in the quantity of the PCR product (vertical axis) in different T cell clones normalized in relation to that of the GAPDH product obtained from the same sample. (B, E) A negative control (lanes −) without added template and a positive control (lanes +) consisting of primers with a cDNA template of an uncloned T. parva -specific CD8 + T cell line (from animal 641) obtained on day 7 after the 3rd stimulation are included. The density of all PCR amplicon bands was measured by Kodak 1D software (version 3.6). The correlation between variables was analyzed by Pearson’s correlation test. P values of

    Techniques Used: Polymerase Chain Reaction, Activity Assay, Clone Assay, Infection, Expressing, Negative Control, Positive Control, Amplification, Software

    (A) 111 In-labeled peptide-pulsed target cells (5 × 10 3 MHC-matched target cells plus Tp1 214–224 at 100 ng/ml) were preincubated with the pan-caspase inhibitor Z-VAD-FMK (80 μM) for 1 h and tested in a 4-h cytotoxicity assay with two Tp1-specific cloned CD8 + T cell lines from animal 641. As controls, effector cells (1 × 10 4 ) preincubated with the perforin inhibitor CMA (10 ng/ml) for 2 h or the granzyme B inhibitor Z-IETD-FMK (40 μM) for 1 h were tested in the same experiment. Labeled target cells alone were also incubated with these inhibitors in the assay. A standard effector-to-target cell ratio of 2:1 was used. (B) Expression vector pET-15b carrying an N-terminal His tag sequence followed by the full-length coding sequence of bovine Bid was expressed in E. coli BL21(DE3) in the presence (lane +) or absence (lane −) of IPTG, and the expressed products were purified using automated immobilized metal affinity chromatography (IMAC) and automated ion-exchange chromatography (IEC). The products were separated by SDS-PAGE and visualized by Coomassie blue staining. The predicted size of bovine recombinant Bid is 23.7 kDa. (C, D) Purified recombinant bovine Bid proteins (3 μg) were incubated with the indicated concentrations of active bovine granzyme B for 2 h at 37°C. (C) The reaction products were separated by SDS-PAGE and visualized by Coomassie blue staining. (D) Full-length recombinant Bid and truncated Bid (N terminus) were detected by anti-His tag antibody, and recombinant granzyme B was detected by anti-FLAG M2 antibody in a Western blot. Inactive bovine granzyme B mutant cells (with an alanine substitution at position 195), mock-transfected cells (pFLAG without an insert), and Cos-7 cells alone were included as negative controls for granzyme B proteolysis specificity.
    Figure Legend Snippet: (A) 111 In-labeled peptide-pulsed target cells (5 × 10 3 MHC-matched target cells plus Tp1 214–224 at 100 ng/ml) were preincubated with the pan-caspase inhibitor Z-VAD-FMK (80 μM) for 1 h and tested in a 4-h cytotoxicity assay with two Tp1-specific cloned CD8 + T cell lines from animal 641. As controls, effector cells (1 × 10 4 ) preincubated with the perforin inhibitor CMA (10 ng/ml) for 2 h or the granzyme B inhibitor Z-IETD-FMK (40 μM) for 1 h were tested in the same experiment. Labeled target cells alone were also incubated with these inhibitors in the assay. A standard effector-to-target cell ratio of 2:1 was used. (B) Expression vector pET-15b carrying an N-terminal His tag sequence followed by the full-length coding sequence of bovine Bid was expressed in E. coli BL21(DE3) in the presence (lane +) or absence (lane −) of IPTG, and the expressed products were purified using automated immobilized metal affinity chromatography (IMAC) and automated ion-exchange chromatography (IEC). The products were separated by SDS-PAGE and visualized by Coomassie blue staining. The predicted size of bovine recombinant Bid is 23.7 kDa. (C, D) Purified recombinant bovine Bid proteins (3 μg) were incubated with the indicated concentrations of active bovine granzyme B for 2 h at 37°C. (C) The reaction products were separated by SDS-PAGE and visualized by Coomassie blue staining. (D) Full-length recombinant Bid and truncated Bid (N terminus) were detected by anti-His tag antibody, and recombinant granzyme B was detected by anti-FLAG M2 antibody in a Western blot. Inactive bovine granzyme B mutant cells (with an alanine substitution at position 195), mock-transfected cells (pFLAG without an insert), and Cos-7 cells alone were included as negative controls for granzyme B proteolysis specificity.

    Techniques Used: Labeling, Cytotoxicity Assay, Clone Assay, Incubation, Expressing, Plasmid Preparation, Positron Emission Tomography, Sequencing, Purification, Affinity Chromatography, Ion Exchange Chromatography, SDS Page, Staining, Recombinant, Western Blot, Mutagenesis, Transfection

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    Article Snippet: .. When LipoK and M. leprae stimulated DCs were co-cultured with T cells, 12.7% of CD4high T cells produced perforin and 14.6% of those cells produced granzyme B, whereas in presence of M. leprae stimulated DCs, 6.6% produced perforin and 8.3% produced granzyme B ( ). .. These data indicated that in addition to CD8+ T cells, CD4+ T cells also had the capacity to produce significant amounts of perforin and granzyme B.

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    Article Snippet: .. For intracellular staining of IFN-γ and granzyme B, brefeldin A (10 μg/ml) (Sigma-Aldrich) was added for the last 4 h prior to acquisition and the IntraStain Kit (Dako Cytomation, Denmark) was used. .. Acquisition was performed on a FACSCanto flow cytometer (BD) and analyzed using the Infinicyt software.

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    Article Snippet: .. For IFN-γ, Granzyme B or Granzyme A staining, cells were stimulated for 4 h with 50ng/ml phorbol 12-myristate 13-acetate (PMA, Sigma) and 1μg/ml ionomycin (Sigma) in the presence of 10μg/ml Brefeldin A. .. After stimulation, cells were stained for antibodies to surface marker, followed by fixation permeabilization with Fixation and Permeabilization buffer (ebioscience) according to the manufacturer’s instructions.

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    Article Title: A Lipopeptide Facilitate Induction of Mycobacterium leprae Killing in Host Cells
    Article Snippet: Together, the results indicate that LipoK could contribute to protective host response against leprosy and eventually kill the bacteria, through the production of perforin, granulysin and granzyme B in T cells.

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    Millipore granzyme b substrate acetyl
    (A) Cytotoxic activity and the levels of <t>granzyme</t> B content and release of 12 T. parva -specific CD8 + T cell clones isolated from two animals (animals 641 and 633) were assayed with autologous T. parva -infected target cells. A standard effector-to-target cell ratio of 2:1 was used. (B, C) Correlation of granzyme B cellular activity with the levels of granzyme B released following antigenic stimulation ( r = 0.953, P
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    (A) Cytotoxic activity and the levels of <t>granzyme</t> B content and release of 12 T. parva -specific CD8 + T cell clones isolated from two animals (animals 641 and 633) were assayed with autologous T. parva -infected target cells. A standard effector-to-target cell ratio of 2:1 was used. (B, C) Correlation of granzyme B cellular activity with the levels of granzyme B released following antigenic stimulation ( r = 0.953, P
    N Acetyl Ile Glu Thr Asp P Nitroanilide, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    (A) Cytotoxic activity and the levels of granzyme B content and release of 12 T. parva -specific CD8 + T cell clones isolated from two animals (animals 641 and 633) were assayed with autologous T. parva -infected target cells. A standard effector-to-target cell ratio of 2:1 was used. (B, C) Correlation of granzyme B cellular activity with the levels of granzyme B released following antigenic stimulation ( r = 0.953, P

    Journal: Infection and Immunity

    Article Title: Granzyme B Is an Essential Mediator in CD8+ T Cell Killing of Theileria parva-Infected Cells

    doi: 10.1128/IAI.00386-18

    Figure Lengend Snippet: (A) Cytotoxic activity and the levels of granzyme B content and release of 12 T. parva -specific CD8 + T cell clones isolated from two animals (animals 641 and 633) were assayed with autologous T. parva -infected target cells. A standard effector-to-target cell ratio of 2:1 was used. (B, C) Correlation of granzyme B cellular activity with the levels of granzyme B released following antigenic stimulation ( r = 0.953, P

    Article Snippet: Supernatants were harvested and assayed in duplicate for protease activity; aliquots of 25 μl of lysis supernatant, the granzyme B substrate acetyl (Ac)–IEPD–p -nitroaniline (p NA) (Calbiochem) at a final concentration of 300 μM, and reaction buffer (0.1 M HEPES, pH 7.0, 0.3 M NaCl, 1 mM EDTA) in a total volume of 250 μl/well were added into the wells of Falcon 96-well flat-bottomed microplates (BD).

    Techniques: Activity Assay, Clone Assay, Isolation, Infection

    (A) Amino acid sequences from the nucleotide sequences of three recombinant forms of bovine granzyme B cDNA aligned with the reference sequence from the genome database. Granzyme B-CDs, the full-length cDNA from the bovine genome assembly, UMD3.1 (accession number corrected_ENSBTAG00000010057); Granzyme B-WT, the pFLAG-CMV-5a vector containing wild-type granzyme B; Granzyme B-Function, the pFLAG-CMV-5a vector containing functional granzyme B; Granzyme B-Mutant, the pFLAG-CMV-5a vector containing functional granzyme B with a Ser 195 -to-Ala 195 mutation; dots, identical residues; dashes, gaps; red box, leader peptide; yellow box, dipeptide/GE; black box, Ser195Ala; blue box, FLAG epitope tag sequence of the pFLAG-CMV-5a vector. (B) Enzymatic activity of different recombinant forms of bovine granzyme B tested on a granzyme B-specific substrate, Ac-IEPD- p NA (filled bars), and a control substrate, Suc-GGF- p NA (empty bars). Cos-7 cells were transiently transfected with unmodified granzyme B cDNA (WT), cDNA with the GE dipeptide deleted (Function), or cDNA containing a deletion of the dipeptide and an alanine substitution at position 195 (Mutant). The transfection efficiencies of Cos-7 cells with the three granzyme B constructs were 35%, 33%, and 33%, respectively. Lysates of the transfected cells collected after 48 h were incubated with the substrates for 4 h. Controls consisted of lysates of cells transfected with pFLAG without an insert (Mock) and buffer (No cells) added to the substrate. The color reaction generated after 4 h by cleavage of the p NA substrate was measured at a wavelength of 405 nm using a Synergy HT multimode microplate reader (BioTek). (C) Inhibition of the functional recombinant cattle granzyme B by preincubating with 10 μM granzyme B-specific inhibitor Ac-IEPD-CHO for 0.5 h.

    Journal: Infection and Immunity

    Article Title: Granzyme B Is an Essential Mediator in CD8+ T Cell Killing of Theileria parva-Infected Cells

    doi: 10.1128/IAI.00386-18

    Figure Lengend Snippet: (A) Amino acid sequences from the nucleotide sequences of three recombinant forms of bovine granzyme B cDNA aligned with the reference sequence from the genome database. Granzyme B-CDs, the full-length cDNA from the bovine genome assembly, UMD3.1 (accession number corrected_ENSBTAG00000010057); Granzyme B-WT, the pFLAG-CMV-5a vector containing wild-type granzyme B; Granzyme B-Function, the pFLAG-CMV-5a vector containing functional granzyme B; Granzyme B-Mutant, the pFLAG-CMV-5a vector containing functional granzyme B with a Ser 195 -to-Ala 195 mutation; dots, identical residues; dashes, gaps; red box, leader peptide; yellow box, dipeptide/GE; black box, Ser195Ala; blue box, FLAG epitope tag sequence of the pFLAG-CMV-5a vector. (B) Enzymatic activity of different recombinant forms of bovine granzyme B tested on a granzyme B-specific substrate, Ac-IEPD- p NA (filled bars), and a control substrate, Suc-GGF- p NA (empty bars). Cos-7 cells were transiently transfected with unmodified granzyme B cDNA (WT), cDNA with the GE dipeptide deleted (Function), or cDNA containing a deletion of the dipeptide and an alanine substitution at position 195 (Mutant). The transfection efficiencies of Cos-7 cells with the three granzyme B constructs were 35%, 33%, and 33%, respectively. Lysates of the transfected cells collected after 48 h were incubated with the substrates for 4 h. Controls consisted of lysates of cells transfected with pFLAG without an insert (Mock) and buffer (No cells) added to the substrate. The color reaction generated after 4 h by cleavage of the p NA substrate was measured at a wavelength of 405 nm using a Synergy HT multimode microplate reader (BioTek). (C) Inhibition of the functional recombinant cattle granzyme B by preincubating with 10 μM granzyme B-specific inhibitor Ac-IEPD-CHO for 0.5 h.

    Article Snippet: Supernatants were harvested and assayed in duplicate for protease activity; aliquots of 25 μl of lysis supernatant, the granzyme B substrate acetyl (Ac)–IEPD–p -nitroaniline (p NA) (Calbiochem) at a final concentration of 300 μM, and reaction buffer (0.1 M HEPES, pH 7.0, 0.3 M NaCl, 1 mM EDTA) in a total volume of 250 μl/well were added into the wells of Falcon 96-well flat-bottomed microplates (BD).

    Techniques: Recombinant, Sequencing, Plasmid Preparation, Functional Assay, Mutagenesis, FLAG-tag, Activity Assay, Transfection, Construct, Incubation, Generated, Inhibition

    Inhibition of the cytotoxic activity of an uncloned (bulk) CD8 + T cell line from animal 011 (A) and three CD8 + T cell lines from animal 592 (B) by incubation with the perforin inhibitor concanamycin A (CMA) and three CD8 + T cell lines from animal 641 by incubation with the granzyme B inhibitor Z-IETD-FMK (C). (A, B) Effectors (1 × 10 4 ) were preincubated with various concentrations of CMA for 2 h and tested in a 4-h cytotoxicity assay with 111 In-labeled autologous TpM target cells and MHC-matched target cells pulsed with a peptide consisting of T. parva antigen Tp2 from residues 49 to 59 (Tp2 49–59 ) (1,000 ng/ml). (C) Three cloned CD8 + T cell lines (1 × 10 4 ) were preincubated for 1 h with 40 μM Z-IETD-FMK and a negative control, Z-VAD-FMK. Labeled target cells alone were also incubated with the inhibitors in the assay. A standard effector-to-target cell ratio of 2:1 was used.

    Journal: Infection and Immunity

    Article Title: Granzyme B Is an Essential Mediator in CD8+ T Cell Killing of Theileria parva-Infected Cells

    doi: 10.1128/IAI.00386-18

    Figure Lengend Snippet: Inhibition of the cytotoxic activity of an uncloned (bulk) CD8 + T cell line from animal 011 (A) and three CD8 + T cell lines from animal 592 (B) by incubation with the perforin inhibitor concanamycin A (CMA) and three CD8 + T cell lines from animal 641 by incubation with the granzyme B inhibitor Z-IETD-FMK (C). (A, B) Effectors (1 × 10 4 ) were preincubated with various concentrations of CMA for 2 h and tested in a 4-h cytotoxicity assay with 111 In-labeled autologous TpM target cells and MHC-matched target cells pulsed with a peptide consisting of T. parva antigen Tp2 from residues 49 to 59 (Tp2 49–59 ) (1,000 ng/ml). (C) Three cloned CD8 + T cell lines (1 × 10 4 ) were preincubated for 1 h with 40 μM Z-IETD-FMK and a negative control, Z-VAD-FMK. Labeled target cells alone were also incubated with the inhibitors in the assay. A standard effector-to-target cell ratio of 2:1 was used.

    Article Snippet: Supernatants were harvested and assayed in duplicate for protease activity; aliquots of 25 μl of lysis supernatant, the granzyme B substrate acetyl (Ac)–IEPD–p -nitroaniline (p NA) (Calbiochem) at a final concentration of 300 μM, and reaction buffer (0.1 M HEPES, pH 7.0, 0.3 M NaCl, 1 mM EDTA) in a total volume of 250 μl/well were added into the wells of Falcon 96-well flat-bottomed microplates (BD).

    Techniques: Inhibition, Activity Assay, Incubation, Cytotoxicity Assay, Labeling, Clone Assay, Negative Control

    (A) PCR products obtained for each of the bovine granule enzymes from an uncloned T. parva -specific CD8 + T cell line (from animal 641). The sizes of the PCR products obtained were as follows: granzyme A (lane A), 838 bp; granzyme O (lane O), 849 bp; granzyme B (lane B), 818 bp; granzyme H (lane H), 820 bp; granzyme K (lane K), 889 bp; granzyme M (lane M), 833 bp; perforin (lane PFN), 1,275 bp. Negative controls (primers with no added cDNA template) were included in the lane to the left. (B) Agarose gels showing the PCR products for granzyme B (457 bp), perforin (1,275 bp), and the GAPDH control (304 bp). The numbers of days after antigenic stimulation are shown. (C) Changes in the quantity of the PCR product (vertical axis) at different times following antigenic stimulation, normalized in relation to that of the GAPDH product obtained from the same sample. (D) Cytotoxic activity of 8 T. parva -specific CD8 + T cell clones from two different animals (animals 641 and 011) assayed on autologous T. parva -infected targets. (E) Agarose gels showing the PCR products for granzyme B (457 bp), perforin (1,275 bp), and the GAPDH control (304 bp) from 8 T. parva -specific CD8 + T cell clones (D). (F) Correlation of killing of Theileria- infected target cells by CD8 + T cell clones with the levels of mRNA expression of granzyme B ( r = 0.438, P = 0.278) and perforin ( r = −0.104, P = 0.806). Changes in the quantity of the PCR product (vertical axis) in different T cell clones normalized in relation to that of the GAPDH product obtained from the same sample. (B, E) A negative control (lanes −) without added template and a positive control (lanes +) consisting of primers with a cDNA template of an uncloned T. parva -specific CD8 + T cell line (from animal 641) obtained on day 7 after the 3rd stimulation are included. The density of all PCR amplicon bands was measured by Kodak 1D software (version 3.6). The correlation between variables was analyzed by Pearson’s correlation test. P values of

    Journal: Infection and Immunity

    Article Title: Granzyme B Is an Essential Mediator in CD8+ T Cell Killing of Theileria parva-Infected Cells

    doi: 10.1128/IAI.00386-18

    Figure Lengend Snippet: (A) PCR products obtained for each of the bovine granule enzymes from an uncloned T. parva -specific CD8 + T cell line (from animal 641). The sizes of the PCR products obtained were as follows: granzyme A (lane A), 838 bp; granzyme O (lane O), 849 bp; granzyme B (lane B), 818 bp; granzyme H (lane H), 820 bp; granzyme K (lane K), 889 bp; granzyme M (lane M), 833 bp; perforin (lane PFN), 1,275 bp. Negative controls (primers with no added cDNA template) were included in the lane to the left. (B) Agarose gels showing the PCR products for granzyme B (457 bp), perforin (1,275 bp), and the GAPDH control (304 bp). The numbers of days after antigenic stimulation are shown. (C) Changes in the quantity of the PCR product (vertical axis) at different times following antigenic stimulation, normalized in relation to that of the GAPDH product obtained from the same sample. (D) Cytotoxic activity of 8 T. parva -specific CD8 + T cell clones from two different animals (animals 641 and 011) assayed on autologous T. parva -infected targets. (E) Agarose gels showing the PCR products for granzyme B (457 bp), perforin (1,275 bp), and the GAPDH control (304 bp) from 8 T. parva -specific CD8 + T cell clones (D). (F) Correlation of killing of Theileria- infected target cells by CD8 + T cell clones with the levels of mRNA expression of granzyme B ( r = 0.438, P = 0.278) and perforin ( r = −0.104, P = 0.806). Changes in the quantity of the PCR product (vertical axis) in different T cell clones normalized in relation to that of the GAPDH product obtained from the same sample. (B, E) A negative control (lanes −) without added template and a positive control (lanes +) consisting of primers with a cDNA template of an uncloned T. parva -specific CD8 + T cell line (from animal 641) obtained on day 7 after the 3rd stimulation are included. The density of all PCR amplicon bands was measured by Kodak 1D software (version 3.6). The correlation between variables was analyzed by Pearson’s correlation test. P values of

    Article Snippet: Supernatants were harvested and assayed in duplicate for protease activity; aliquots of 25 μl of lysis supernatant, the granzyme B substrate acetyl (Ac)–IEPD–p -nitroaniline (p NA) (Calbiochem) at a final concentration of 300 μM, and reaction buffer (0.1 M HEPES, pH 7.0, 0.3 M NaCl, 1 mM EDTA) in a total volume of 250 μl/well were added into the wells of Falcon 96-well flat-bottomed microplates (BD).

    Techniques: Polymerase Chain Reaction, Activity Assay, Clone Assay, Infection, Expressing, Negative Control, Positive Control, Amplification, Software

    (A) 111 In-labeled peptide-pulsed target cells (5 × 10 3 MHC-matched target cells plus Tp1 214–224 at 100 ng/ml) were preincubated with the pan-caspase inhibitor Z-VAD-FMK (80 μM) for 1 h and tested in a 4-h cytotoxicity assay with two Tp1-specific cloned CD8 + T cell lines from animal 641. As controls, effector cells (1 × 10 4 ) preincubated with the perforin inhibitor CMA (10 ng/ml) for 2 h or the granzyme B inhibitor Z-IETD-FMK (40 μM) for 1 h were tested in the same experiment. Labeled target cells alone were also incubated with these inhibitors in the assay. A standard effector-to-target cell ratio of 2:1 was used. (B) Expression vector pET-15b carrying an N-terminal His tag sequence followed by the full-length coding sequence of bovine Bid was expressed in E. coli BL21(DE3) in the presence (lane +) or absence (lane −) of IPTG, and the expressed products were purified using automated immobilized metal affinity chromatography (IMAC) and automated ion-exchange chromatography (IEC). The products were separated by SDS-PAGE and visualized by Coomassie blue staining. The predicted size of bovine recombinant Bid is 23.7 kDa. (C, D) Purified recombinant bovine Bid proteins (3 μg) were incubated with the indicated concentrations of active bovine granzyme B for 2 h at 37°C. (C) The reaction products were separated by SDS-PAGE and visualized by Coomassie blue staining. (D) Full-length recombinant Bid and truncated Bid (N terminus) were detected by anti-His tag antibody, and recombinant granzyme B was detected by anti-FLAG M2 antibody in a Western blot. Inactive bovine granzyme B mutant cells (with an alanine substitution at position 195), mock-transfected cells (pFLAG without an insert), and Cos-7 cells alone were included as negative controls for granzyme B proteolysis specificity.

    Journal: Infection and Immunity

    Article Title: Granzyme B Is an Essential Mediator in CD8+ T Cell Killing of Theileria parva-Infected Cells

    doi: 10.1128/IAI.00386-18

    Figure Lengend Snippet: (A) 111 In-labeled peptide-pulsed target cells (5 × 10 3 MHC-matched target cells plus Tp1 214–224 at 100 ng/ml) were preincubated with the pan-caspase inhibitor Z-VAD-FMK (80 μM) for 1 h and tested in a 4-h cytotoxicity assay with two Tp1-specific cloned CD8 + T cell lines from animal 641. As controls, effector cells (1 × 10 4 ) preincubated with the perforin inhibitor CMA (10 ng/ml) for 2 h or the granzyme B inhibitor Z-IETD-FMK (40 μM) for 1 h were tested in the same experiment. Labeled target cells alone were also incubated with these inhibitors in the assay. A standard effector-to-target cell ratio of 2:1 was used. (B) Expression vector pET-15b carrying an N-terminal His tag sequence followed by the full-length coding sequence of bovine Bid was expressed in E. coli BL21(DE3) in the presence (lane +) or absence (lane −) of IPTG, and the expressed products were purified using automated immobilized metal affinity chromatography (IMAC) and automated ion-exchange chromatography (IEC). The products were separated by SDS-PAGE and visualized by Coomassie blue staining. The predicted size of bovine recombinant Bid is 23.7 kDa. (C, D) Purified recombinant bovine Bid proteins (3 μg) were incubated with the indicated concentrations of active bovine granzyme B for 2 h at 37°C. (C) The reaction products were separated by SDS-PAGE and visualized by Coomassie blue staining. (D) Full-length recombinant Bid and truncated Bid (N terminus) were detected by anti-His tag antibody, and recombinant granzyme B was detected by anti-FLAG M2 antibody in a Western blot. Inactive bovine granzyme B mutant cells (with an alanine substitution at position 195), mock-transfected cells (pFLAG without an insert), and Cos-7 cells alone were included as negative controls for granzyme B proteolysis specificity.

    Article Snippet: Supernatants were harvested and assayed in duplicate for protease activity; aliquots of 25 μl of lysis supernatant, the granzyme B substrate acetyl (Ac)–IEPD–p -nitroaniline (p NA) (Calbiochem) at a final concentration of 300 μM, and reaction buffer (0.1 M HEPES, pH 7.0, 0.3 M NaCl, 1 mM EDTA) in a total volume of 250 μl/well were added into the wells of Falcon 96-well flat-bottomed microplates (BD).

    Techniques: Labeling, Cytotoxicity Assay, Clone Assay, Incubation, Expressing, Plasmid Preparation, Positron Emission Tomography, Sequencing, Purification, Affinity Chromatography, Ion Exchange Chromatography, SDS Page, Staining, Recombinant, Western Blot, Mutagenesis, Transfection

    (A) Cytotoxic activity and the levels of granzyme B content and release of 12 T. parva -specific CD8 + T cell clones isolated from two animals (animals 641 and 633) were assayed with autologous T. parva -infected target cells. A standard effector-to-target cell ratio of 2:1 was used. (B, C) Correlation of granzyme B cellular activity with the levels of granzyme B released following antigenic stimulation ( r = 0.953, P

    Journal: Infection and Immunity

    Article Title: Granzyme B Is an Essential Mediator in CD8+ T Cell Killing of Theileria parva-Infected Cells

    doi: 10.1128/IAI.00386-18

    Figure Lengend Snippet: (A) Cytotoxic activity and the levels of granzyme B content and release of 12 T. parva -specific CD8 + T cell clones isolated from two animals (animals 641 and 633) were assayed with autologous T. parva -infected target cells. A standard effector-to-target cell ratio of 2:1 was used. (B, C) Correlation of granzyme B cellular activity with the levels of granzyme B released following antigenic stimulation ( r = 0.953, P

    Article Snippet: Supernatants were harvested and assayed in duplicate for protease activity; aliquots of 25 μl of lysis supernatant, the granzyme B substrate acetyl (Ac)–IEPD– p -nitroaniline ( p NA) (Calbiochem) at a final concentration of 300 μM, and reaction buffer (0.1 M HEPES, pH 7.0, 0.3 M NaCl, 1 mM EDTA) in a total volume of 250 μl/well were added into the wells of Falcon 96-well flat-bottomed microplates (BD).

    Techniques: Activity Assay, Clone Assay, Isolation, Infection

    (A) Amino acid sequences from the nucleotide sequences of three recombinant forms of bovine granzyme B cDNA aligned with the reference sequence from the genome database. Granzyme B-CDs, the full-length cDNA from the bovine genome assembly, UMD3.1 (accession number corrected_ENSBTAG00000010057); Granzyme B-WT, the pFLAG-CMV-5a vector containing wild-type granzyme B; Granzyme B-Function, the pFLAG-CMV-5a vector containing functional granzyme B; Granzyme B-Mutant, the pFLAG-CMV-5a vector containing functional granzyme B with a Ser 195 -to-Ala 195 mutation; dots, identical residues; dashes, gaps; red box, leader peptide; yellow box, dipeptide/GE; black box, Ser195Ala; blue box, FLAG epitope tag sequence of the pFLAG-CMV-5a vector. (B) Enzymatic activity of different recombinant forms of bovine granzyme B tested on a granzyme B-specific substrate, Ac-IEPD- p NA (filled bars), and a control substrate, Suc-GGF- p NA (empty bars). Cos-7 cells were transiently transfected with unmodified granzyme B cDNA (WT), cDNA with the GE dipeptide deleted (Function), or cDNA containing a deletion of the dipeptide and an alanine substitution at position 195 (Mutant). The transfection efficiencies of Cos-7 cells with the three granzyme B constructs were 35%, 33%, and 33%, respectively. Lysates of the transfected cells collected after 48 h were incubated with the substrates for 4 h. Controls consisted of lysates of cells transfected with pFLAG without an insert (Mock) and buffer (No cells) added to the substrate. The color reaction generated after 4 h by cleavage of the p NA substrate was measured at a wavelength of 405 nm using a Synergy HT multimode microplate reader (BioTek). (C) Inhibition of the functional recombinant cattle granzyme B by preincubating with 10 μM granzyme B-specific inhibitor Ac-IEPD-CHO for 0.5 h.

    Journal: Infection and Immunity

    Article Title: Granzyme B Is an Essential Mediator in CD8+ T Cell Killing of Theileria parva-Infected Cells

    doi: 10.1128/IAI.00386-18

    Figure Lengend Snippet: (A) Amino acid sequences from the nucleotide sequences of three recombinant forms of bovine granzyme B cDNA aligned with the reference sequence from the genome database. Granzyme B-CDs, the full-length cDNA from the bovine genome assembly, UMD3.1 (accession number corrected_ENSBTAG00000010057); Granzyme B-WT, the pFLAG-CMV-5a vector containing wild-type granzyme B; Granzyme B-Function, the pFLAG-CMV-5a vector containing functional granzyme B; Granzyme B-Mutant, the pFLAG-CMV-5a vector containing functional granzyme B with a Ser 195 -to-Ala 195 mutation; dots, identical residues; dashes, gaps; red box, leader peptide; yellow box, dipeptide/GE; black box, Ser195Ala; blue box, FLAG epitope tag sequence of the pFLAG-CMV-5a vector. (B) Enzymatic activity of different recombinant forms of bovine granzyme B tested on a granzyme B-specific substrate, Ac-IEPD- p NA (filled bars), and a control substrate, Suc-GGF- p NA (empty bars). Cos-7 cells were transiently transfected with unmodified granzyme B cDNA (WT), cDNA with the GE dipeptide deleted (Function), or cDNA containing a deletion of the dipeptide and an alanine substitution at position 195 (Mutant). The transfection efficiencies of Cos-7 cells with the three granzyme B constructs were 35%, 33%, and 33%, respectively. Lysates of the transfected cells collected after 48 h were incubated with the substrates for 4 h. Controls consisted of lysates of cells transfected with pFLAG without an insert (Mock) and buffer (No cells) added to the substrate. The color reaction generated after 4 h by cleavage of the p NA substrate was measured at a wavelength of 405 nm using a Synergy HT multimode microplate reader (BioTek). (C) Inhibition of the functional recombinant cattle granzyme B by preincubating with 10 μM granzyme B-specific inhibitor Ac-IEPD-CHO for 0.5 h.

    Article Snippet: Supernatants were harvested and assayed in duplicate for protease activity; aliquots of 25 μl of lysis supernatant, the granzyme B substrate acetyl (Ac)–IEPD– p -nitroaniline ( p NA) (Calbiochem) at a final concentration of 300 μM, and reaction buffer (0.1 M HEPES, pH 7.0, 0.3 M NaCl, 1 mM EDTA) in a total volume of 250 μl/well were added into the wells of Falcon 96-well flat-bottomed microplates (BD).

    Techniques: Recombinant, Sequencing, Plasmid Preparation, Functional Assay, Mutagenesis, FLAG-tag, Activity Assay, Transfection, Construct, Incubation, Generated, Inhibition

    Inhibition of the cytotoxic activity of an uncloned (bulk) CD8 + T cell line from animal 011 (A) and three CD8 + T cell lines from animal 592 (B) by incubation with the perforin inhibitor concanamycin A (CMA) and three CD8 + T cell lines from animal 641 by incubation with the granzyme B inhibitor Z-IETD-FMK (C). (A, B) Effectors (1 × 10 4 ) were preincubated with various concentrations of CMA for 2 h and tested in a 4-h cytotoxicity assay with 111 In-labeled autologous TpM target cells and MHC-matched target cells pulsed with a peptide consisting of T. parva antigen Tp2 from residues 49 to 59 (Tp2 49–59 ) (1,000 ng/ml). (C) Three cloned CD8 + T cell lines (1 × 10 4 ) were preincubated for 1 h with 40 μM Z-IETD-FMK and a negative control, Z-VAD-FMK. Labeled target cells alone were also incubated with the inhibitors in the assay. A standard effector-to-target cell ratio of 2:1 was used.

    Journal: Infection and Immunity

    Article Title: Granzyme B Is an Essential Mediator in CD8+ T Cell Killing of Theileria parva-Infected Cells

    doi: 10.1128/IAI.00386-18

    Figure Lengend Snippet: Inhibition of the cytotoxic activity of an uncloned (bulk) CD8 + T cell line from animal 011 (A) and three CD8 + T cell lines from animal 592 (B) by incubation with the perforin inhibitor concanamycin A (CMA) and three CD8 + T cell lines from animal 641 by incubation with the granzyme B inhibitor Z-IETD-FMK (C). (A, B) Effectors (1 × 10 4 ) were preincubated with various concentrations of CMA for 2 h and tested in a 4-h cytotoxicity assay with 111 In-labeled autologous TpM target cells and MHC-matched target cells pulsed with a peptide consisting of T. parva antigen Tp2 from residues 49 to 59 (Tp2 49–59 ) (1,000 ng/ml). (C) Three cloned CD8 + T cell lines (1 × 10 4 ) were preincubated for 1 h with 40 μM Z-IETD-FMK and a negative control, Z-VAD-FMK. Labeled target cells alone were also incubated with the inhibitors in the assay. A standard effector-to-target cell ratio of 2:1 was used.

    Article Snippet: Supernatants were harvested and assayed in duplicate for protease activity; aliquots of 25 μl of lysis supernatant, the granzyme B substrate acetyl (Ac)–IEPD– p -nitroaniline ( p NA) (Calbiochem) at a final concentration of 300 μM, and reaction buffer (0.1 M HEPES, pH 7.0, 0.3 M NaCl, 1 mM EDTA) in a total volume of 250 μl/well were added into the wells of Falcon 96-well flat-bottomed microplates (BD).

    Techniques: Inhibition, Activity Assay, Incubation, Cytotoxicity Assay, Labeling, Clone Assay, Negative Control

    (A) PCR products obtained for each of the bovine granule enzymes from an uncloned T. parva -specific CD8 + T cell line (from animal 641). The sizes of the PCR products obtained were as follows: granzyme A (lane A), 838 bp; granzyme O (lane O), 849 bp; granzyme B (lane B), 818 bp; granzyme H (lane H), 820 bp; granzyme K (lane K), 889 bp; granzyme M (lane M), 833 bp; perforin (lane PFN), 1,275 bp. Negative controls (primers with no added cDNA template) were included in the lane to the left. (B) Agarose gels showing the PCR products for granzyme B (457 bp), perforin (1,275 bp), and the GAPDH control (304 bp). The numbers of days after antigenic stimulation are shown. (C) Changes in the quantity of the PCR product (vertical axis) at different times following antigenic stimulation, normalized in relation to that of the GAPDH product obtained from the same sample. (D) Cytotoxic activity of 8 T. parva -specific CD8 + T cell clones from two different animals (animals 641 and 011) assayed on autologous T. parva -infected targets. (E) Agarose gels showing the PCR products for granzyme B (457 bp), perforin (1,275 bp), and the GAPDH control (304 bp) from 8 T. parva -specific CD8 + T cell clones (D). (F) Correlation of killing of Theileria- infected target cells by CD8 + T cell clones with the levels of mRNA expression of granzyme B ( r = 0.438, P = 0.278) and perforin ( r = −0.104, P = 0.806). Changes in the quantity of the PCR product (vertical axis) in different T cell clones normalized in relation to that of the GAPDH product obtained from the same sample. (B, E) A negative control (lanes −) without added template and a positive control (lanes +) consisting of primers with a cDNA template of an uncloned T. parva -specific CD8 + T cell line (from animal 641) obtained on day 7 after the 3rd stimulation are included. The density of all PCR amplicon bands was measured by Kodak 1D software (version 3.6). The correlation between variables was analyzed by Pearson’s correlation test. P values of

    Journal: Infection and Immunity

    Article Title: Granzyme B Is an Essential Mediator in CD8+ T Cell Killing of Theileria parva-Infected Cells

    doi: 10.1128/IAI.00386-18

    Figure Lengend Snippet: (A) PCR products obtained for each of the bovine granule enzymes from an uncloned T. parva -specific CD8 + T cell line (from animal 641). The sizes of the PCR products obtained were as follows: granzyme A (lane A), 838 bp; granzyme O (lane O), 849 bp; granzyme B (lane B), 818 bp; granzyme H (lane H), 820 bp; granzyme K (lane K), 889 bp; granzyme M (lane M), 833 bp; perforin (lane PFN), 1,275 bp. Negative controls (primers with no added cDNA template) were included in the lane to the left. (B) Agarose gels showing the PCR products for granzyme B (457 bp), perforin (1,275 bp), and the GAPDH control (304 bp). The numbers of days after antigenic stimulation are shown. (C) Changes in the quantity of the PCR product (vertical axis) at different times following antigenic stimulation, normalized in relation to that of the GAPDH product obtained from the same sample. (D) Cytotoxic activity of 8 T. parva -specific CD8 + T cell clones from two different animals (animals 641 and 011) assayed on autologous T. parva -infected targets. (E) Agarose gels showing the PCR products for granzyme B (457 bp), perforin (1,275 bp), and the GAPDH control (304 bp) from 8 T. parva -specific CD8 + T cell clones (D). (F) Correlation of killing of Theileria- infected target cells by CD8 + T cell clones with the levels of mRNA expression of granzyme B ( r = 0.438, P = 0.278) and perforin ( r = −0.104, P = 0.806). Changes in the quantity of the PCR product (vertical axis) in different T cell clones normalized in relation to that of the GAPDH product obtained from the same sample. (B, E) A negative control (lanes −) without added template and a positive control (lanes +) consisting of primers with a cDNA template of an uncloned T. parva -specific CD8 + T cell line (from animal 641) obtained on day 7 after the 3rd stimulation are included. The density of all PCR amplicon bands was measured by Kodak 1D software (version 3.6). The correlation between variables was analyzed by Pearson’s correlation test. P values of

    Article Snippet: Supernatants were harvested and assayed in duplicate for protease activity; aliquots of 25 μl of lysis supernatant, the granzyme B substrate acetyl (Ac)–IEPD– p -nitroaniline ( p NA) (Calbiochem) at a final concentration of 300 μM, and reaction buffer (0.1 M HEPES, pH 7.0, 0.3 M NaCl, 1 mM EDTA) in a total volume of 250 μl/well were added into the wells of Falcon 96-well flat-bottomed microplates (BD).

    Techniques: Polymerase Chain Reaction, Activity Assay, Clone Assay, Infection, Expressing, Negative Control, Positive Control, Amplification, Software

    (A) 111 In-labeled peptide-pulsed target cells (5 × 10 3 MHC-matched target cells plus Tp1 214–224 at 100 ng/ml) were preincubated with the pan-caspase inhibitor Z-VAD-FMK (80 μM) for 1 h and tested in a 4-h cytotoxicity assay with two Tp1-specific cloned CD8 + T cell lines from animal 641. As controls, effector cells (1 × 10 4 ) preincubated with the perforin inhibitor CMA (10 ng/ml) for 2 h or the granzyme B inhibitor Z-IETD-FMK (40 μM) for 1 h were tested in the same experiment. Labeled target cells alone were also incubated with these inhibitors in the assay. A standard effector-to-target cell ratio of 2:1 was used. (B) Expression vector pET-15b carrying an N-terminal His tag sequence followed by the full-length coding sequence of bovine Bid was expressed in E. coli BL21(DE3) in the presence (lane +) or absence (lane −) of IPTG, and the expressed products were purified using automated immobilized metal affinity chromatography (IMAC) and automated ion-exchange chromatography (IEC). The products were separated by SDS-PAGE and visualized by Coomassie blue staining. The predicted size of bovine recombinant Bid is 23.7 kDa. (C, D) Purified recombinant bovine Bid proteins (3 μg) were incubated with the indicated concentrations of active bovine granzyme B for 2 h at 37°C. (C) The reaction products were separated by SDS-PAGE and visualized by Coomassie blue staining. (D) Full-length recombinant Bid and truncated Bid (N terminus) were detected by anti-His tag antibody, and recombinant granzyme B was detected by anti-FLAG M2 antibody in a Western blot. Inactive bovine granzyme B mutant cells (with an alanine substitution at position 195), mock-transfected cells (pFLAG without an insert), and Cos-7 cells alone were included as negative controls for granzyme B proteolysis specificity.

    Journal: Infection and Immunity

    Article Title: Granzyme B Is an Essential Mediator in CD8+ T Cell Killing of Theileria parva-Infected Cells

    doi: 10.1128/IAI.00386-18

    Figure Lengend Snippet: (A) 111 In-labeled peptide-pulsed target cells (5 × 10 3 MHC-matched target cells plus Tp1 214–224 at 100 ng/ml) were preincubated with the pan-caspase inhibitor Z-VAD-FMK (80 μM) for 1 h and tested in a 4-h cytotoxicity assay with two Tp1-specific cloned CD8 + T cell lines from animal 641. As controls, effector cells (1 × 10 4 ) preincubated with the perforin inhibitor CMA (10 ng/ml) for 2 h or the granzyme B inhibitor Z-IETD-FMK (40 μM) for 1 h were tested in the same experiment. Labeled target cells alone were also incubated with these inhibitors in the assay. A standard effector-to-target cell ratio of 2:1 was used. (B) Expression vector pET-15b carrying an N-terminal His tag sequence followed by the full-length coding sequence of bovine Bid was expressed in E. coli BL21(DE3) in the presence (lane +) or absence (lane −) of IPTG, and the expressed products were purified using automated immobilized metal affinity chromatography (IMAC) and automated ion-exchange chromatography (IEC). The products were separated by SDS-PAGE and visualized by Coomassie blue staining. The predicted size of bovine recombinant Bid is 23.7 kDa. (C, D) Purified recombinant bovine Bid proteins (3 μg) were incubated with the indicated concentrations of active bovine granzyme B for 2 h at 37°C. (C) The reaction products were separated by SDS-PAGE and visualized by Coomassie blue staining. (D) Full-length recombinant Bid and truncated Bid (N terminus) were detected by anti-His tag antibody, and recombinant granzyme B was detected by anti-FLAG M2 antibody in a Western blot. Inactive bovine granzyme B mutant cells (with an alanine substitution at position 195), mock-transfected cells (pFLAG without an insert), and Cos-7 cells alone were included as negative controls for granzyme B proteolysis specificity.

    Article Snippet: Supernatants were harvested and assayed in duplicate for protease activity; aliquots of 25 μl of lysis supernatant, the granzyme B substrate acetyl (Ac)–IEPD– p -nitroaniline ( p NA) (Calbiochem) at a final concentration of 300 μM, and reaction buffer (0.1 M HEPES, pH 7.0, 0.3 M NaCl, 1 mM EDTA) in a total volume of 250 μl/well were added into the wells of Falcon 96-well flat-bottomed microplates (BD).

    Techniques: Labeling, Cytotoxicity Assay, Clone Assay, Incubation, Expressing, Plasmid Preparation, Positron Emission Tomography, Sequencing, Purification, Affinity Chromatography, Ion Exchange Chromatography, SDS Page, Staining, Recombinant, Western Blot, Mutagenesis, Transfection