proteinase k  (Millipore)


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    Structured Review

    Millipore proteinase k
    DC-derived EV are enriched in mediators with reported chemoattractant properties. ( a ) Chemokines and growth factors enriched/depleted in DC-EV comparatively to DC conditioned media, as assayed by antibody array. Red dashed lines represent the fold-change thresholds of 1.5 and −1.5. Mediators with an absolute fold-change ≥1.5 are highlighted by the green box. See also Supplementary Fig.  S2 . ( b ) Detection of osteopontin (OPN) in the DC-derived EV fraction (DC-EV) and 100 K supernatant (UC control) by western blot. Proteinase K (PK) digestion, before or after vesicle lysis, were performed as indicated. Full gel scan is available in Supplementary Fig.  S5 . Relative concentration (average ± SD) of ( c ) IL-6 and ( d ) MCP-1 in DC-derived EV (after proteinase K digestion) and 100 K supernatant (UC control), as determined by ELISA. For all assays, EV, 100 K supernatant and conditioned media from 3 different DC donors were pooled together.
    Proteinase K, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1524 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Dendritic Cell-derived Extracellular Vesicles mediate Mesenchymal Stem/Stromal Cell recruitment"

    Article Title: Dendritic Cell-derived Extracellular Vesicles mediate Mesenchymal Stem/Stromal Cell recruitment

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-01809-x

    DC-derived EV are enriched in mediators with reported chemoattractant properties. ( a ) Chemokines and growth factors enriched/depleted in DC-EV comparatively to DC conditioned media, as assayed by antibody array. Red dashed lines represent the fold-change thresholds of 1.5 and −1.5. Mediators with an absolute fold-change ≥1.5 are highlighted by the green box. See also Supplementary Fig.  S2 . ( b ) Detection of osteopontin (OPN) in the DC-derived EV fraction (DC-EV) and 100 K supernatant (UC control) by western blot. Proteinase K (PK) digestion, before or after vesicle lysis, were performed as indicated. Full gel scan is available in Supplementary Fig.  S5 . Relative concentration (average ± SD) of ( c ) IL-6 and ( d ) MCP-1 in DC-derived EV (after proteinase K digestion) and 100 K supernatant (UC control), as determined by ELISA. For all assays, EV, 100 K supernatant and conditioned media from 3 different DC donors were pooled together.
    Figure Legend Snippet: DC-derived EV are enriched in mediators with reported chemoattractant properties. ( a ) Chemokines and growth factors enriched/depleted in DC-EV comparatively to DC conditioned media, as assayed by antibody array. Red dashed lines represent the fold-change thresholds of 1.5 and −1.5. Mediators with an absolute fold-change ≥1.5 are highlighted by the green box. See also Supplementary Fig.  S2 . ( b ) Detection of osteopontin (OPN) in the DC-derived EV fraction (DC-EV) and 100 K supernatant (UC control) by western blot. Proteinase K (PK) digestion, before or after vesicle lysis, were performed as indicated. Full gel scan is available in Supplementary Fig.  S5 . Relative concentration (average ± SD) of ( c ) IL-6 and ( d ) MCP-1 in DC-derived EV (after proteinase K digestion) and 100 K supernatant (UC control), as determined by ELISA. For all assays, EV, 100 K supernatant and conditioned media from 3 different DC donors were pooled together.

    Techniques Used: Derivative Assay, Ab Array, Western Blot, Lysis, Concentration Assay, Enzyme-linked Immunosorbent Assay

    DC-derived EV contain MMP-9 capable of promoting MSC migration. ( a ) Detection of MMP-9 and MMP-2 activity by gelatin zymography in media collected from transwell assays. Cell culture media from top and bottom compartments of MSC recruitment transwell assays was collected separately. Protein was precipitated from these media (Presence of MSC) and from DC-EV pellets (Absence of MSC), resolved by gelatin zymography, and proteolytic activity was detected as clear bands on Coomassie Brilliant Blue stained gels. Bands corresponding to the proenzyme and the active form of each MMP are indicated at the corresponding molecular weight next to gels images. ( b ) Representative imaging flow cytometry plots (top panel) and images (bottom panel) of MMP-9 and HLA-DR detection in DC-derived EV coupled to beads. Beads with EV were stained with HLA-DR-FITC antibodies before being permeabilized and stained using anti-MMP-9 followed by Cy3-secondary antibodies, being detected as defined dots in the periphery of the beads. ( c ) Gelatin zymography detection of MMP-9 in DC-derived EV (DC-EV), and the 100 K ultracentrifugation supernatant control (UC control). Proteinase K (PK) digestion, with or without previous vesicle lysis, were performed as indicated. Pro-MMP-9 and active MMP-9 are indicated at the corresponding molecular weight. Full gel scans are available in Supplementary Fig.  S6 .
    Figure Legend Snippet: DC-derived EV contain MMP-9 capable of promoting MSC migration. ( a ) Detection of MMP-9 and MMP-2 activity by gelatin zymography in media collected from transwell assays. Cell culture media from top and bottom compartments of MSC recruitment transwell assays was collected separately. Protein was precipitated from these media (Presence of MSC) and from DC-EV pellets (Absence of MSC), resolved by gelatin zymography, and proteolytic activity was detected as clear bands on Coomassie Brilliant Blue stained gels. Bands corresponding to the proenzyme and the active form of each MMP are indicated at the corresponding molecular weight next to gels images. ( b ) Representative imaging flow cytometry plots (top panel) and images (bottom panel) of MMP-9 and HLA-DR detection in DC-derived EV coupled to beads. Beads with EV were stained with HLA-DR-FITC antibodies before being permeabilized and stained using anti-MMP-9 followed by Cy3-secondary antibodies, being detected as defined dots in the periphery of the beads. ( c ) Gelatin zymography detection of MMP-9 in DC-derived EV (DC-EV), and the 100 K ultracentrifugation supernatant control (UC control). Proteinase K (PK) digestion, with or without previous vesicle lysis, were performed as indicated. Pro-MMP-9 and active MMP-9 are indicated at the corresponding molecular weight. Full gel scans are available in Supplementary Fig.  S6 .

    Techniques Used: Derivative Assay, Migration, Activity Assay, Zymography, Cell Culture, Staining, Molecular Weight, Imaging, Flow Cytometry, Cytometry, Lysis

    2) Product Images from "Glycoprotein 3 of Porcine Reproductive and Respiratory Syndrome Virus Exhibits an Unusual Hairpin-Like Membrane Topology"

    Article Title: Glycoprotein 3 of Porcine Reproductive and Respiratory Syndrome Virus Exhibits an Unusual Hairpin-Like Membrane Topology

    Journal: Journal of Virology

    doi: 10.1128/JVI.00660-18

    C terminus of GP3 is translocated into the lumen of the ER. (A) Primary structure of GP3 with signal peptide (SP), hydrophobic region (HR), six conserved cysteines (lines), and glycosylation sites (branches; numbering of sites is for PRRSV-2 strains). The position of the first site differs between PRRSV-1 and PRRSV-2 strains. Glycosylation site 138 is an additional site present in GP3 from the IAF-Klop strain, and GP3 from XH-GD lacks the site at position 152. The graph shows the percent conservation ( y axis) of amino acids at each position ( x axis) of a consensus sequence compiled from all PRRSV-1 and PRRSV-2 GP3 sequences present in the database. (B) CHO-K1 cells expressing GP3-YFP from 5 different PRRSV strains and (as a control) a type I transmembrane protein (GP4-YFP from equine arteritis virus [EAV]) were treated with digitonin for 1 min and with proteinase K for 66 min. After each time point, the same microscopic field was recorded with an epifluorescence microscope. (C and D) Two additional glycosylation sites were inserted into the C-terminal part of GP3 from VR-2332 (C) or LV (D), and constructs were expressed in BHK cells. Twenty-four h after transfection, cells were lysed and samples subjected to SDS-PAGE and Western blotting with anti-HA antibody. The C-terminal sequences of GP3 (amino acids 227 to 254 in VR-2332 and 228 to 265 in LV) are shown above the blot. Boldface letters indicate the N-glycosylation site inserted by exchange of one amino acid (underlined). The SDS-PAGE mobility of molecular weight makers are indicated at the left side of the blot. Mock, untransfected cells.
    Figure Legend Snippet: C terminus of GP3 is translocated into the lumen of the ER. (A) Primary structure of GP3 with signal peptide (SP), hydrophobic region (HR), six conserved cysteines (lines), and glycosylation sites (branches; numbering of sites is for PRRSV-2 strains). The position of the first site differs between PRRSV-1 and PRRSV-2 strains. Glycosylation site 138 is an additional site present in GP3 from the IAF-Klop strain, and GP3 from XH-GD lacks the site at position 152. The graph shows the percent conservation ( y axis) of amino acids at each position ( x axis) of a consensus sequence compiled from all PRRSV-1 and PRRSV-2 GP3 sequences present in the database. (B) CHO-K1 cells expressing GP3-YFP from 5 different PRRSV strains and (as a control) a type I transmembrane protein (GP4-YFP from equine arteritis virus [EAV]) were treated with digitonin for 1 min and with proteinase K for 66 min. After each time point, the same microscopic field was recorded with an epifluorescence microscope. (C and D) Two additional glycosylation sites were inserted into the C-terminal part of GP3 from VR-2332 (C) or LV (D), and constructs were expressed in BHK cells. Twenty-four h after transfection, cells were lysed and samples subjected to SDS-PAGE and Western blotting with anti-HA antibody. The C-terminal sequences of GP3 (amino acids 227 to 254 in VR-2332 and 228 to 265 in LV) are shown above the blot. Boldface letters indicate the N-glycosylation site inserted by exchange of one amino acid (underlined). The SDS-PAGE mobility of molecular weight makers are indicated at the left side of the blot. Mock, untransfected cells.

    Techniques Used: Sequencing, Expressing, Microscopy, Construct, Transfection, SDS Page, Western Blot, Hemagglutination Assay, Molecular Weight

    3) Product Images from "Early Murine T-lymphocyte Activation Is Accompanied by a Switch from N-Glycolyl- to "

    Article Title: Early Murine T-lymphocyte Activation Is Accompanied by a Switch from N-Glycolyl- to

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.243410

    Activation of murine T-lymphocytes leads to up-regulation in the binding of siglec-E to cell surface proteins in a sialic acid-dependent manner. A , T-lymphocytes were activated with anti-CD3 and anti-CD28 for 24 h and then cultured for up to 4 days in the presence of IL-2. Cells harvested at each time point were incubated with fluorophore-conjugated CD4 ( left panel ) and CD8 antibodies ( right panel ) together with a pre-complex of FITC-conjugated goat anti-human Fc and siglec-Fc. Mean Fluorescence Intensity ( MFI ) of sialic acid-dependent binding by FACS is shown following subtraction of MFI values for sialidase-treated cells. B , for the detection of MAL and SNA binding, harvested cells corresponding to each time point were incubated with biotinylated lectin followed by a secondary incubation with FITC-conjugated streptavidin. C , splenocytes harvested at 24 h were untreated ( open histograms ) or pretreated with V. cholerae sialidase ( left panel ) or proteinase K ( right panel ) (shaded histograms) and labeled with siglec-E-Fc/anti-Fc-FITC complexes. Data are representative of at least three independent experiments.
    Figure Legend Snippet: Activation of murine T-lymphocytes leads to up-regulation in the binding of siglec-E to cell surface proteins in a sialic acid-dependent manner. A , T-lymphocytes were activated with anti-CD3 and anti-CD28 for 24 h and then cultured for up to 4 days in the presence of IL-2. Cells harvested at each time point were incubated with fluorophore-conjugated CD4 ( left panel ) and CD8 antibodies ( right panel ) together with a pre-complex of FITC-conjugated goat anti-human Fc and siglec-Fc. Mean Fluorescence Intensity ( MFI ) of sialic acid-dependent binding by FACS is shown following subtraction of MFI values for sialidase-treated cells. B , for the detection of MAL and SNA binding, harvested cells corresponding to each time point were incubated with biotinylated lectin followed by a secondary incubation with FITC-conjugated streptavidin. C , splenocytes harvested at 24 h were untreated ( open histograms ) or pretreated with V. cholerae sialidase ( left panel ) or proteinase K ( right panel ) (shaded histograms) and labeled with siglec-E-Fc/anti-Fc-FITC complexes. Data are representative of at least three independent experiments.

    Techniques Used: Activation Assay, Binding Assay, Cell Culture, Incubation, Flow Cytometry, Fluorescence, FACS, Labeling

    4) Product Images from "Characterization of extracellular polymeric matrix, and treatment of Fusobacterium nucleatum and Porphyromonas gingivalis biofilms with DNase I and proteinase K"

    Article Title: Characterization of extracellular polymeric matrix, and treatment of Fusobacterium nucleatum and Porphyromonas gingivalis biofilms with DNase I and proteinase K

    Journal: Journal of Oral Microbiology

    doi: 10.3402/jom.v5i0.20015

    Effect of DNase I and proteinase K on the biofilm formation (time zero) and on 48-h-old biofilm. Fusobacterium nucleatum type strain ATCC 25586 (F.n) and Porphyromonas gingivalis type strain ATCC 33277 (P.g 33277) bacterial species were tested when they were grown as monoculture or as dual species culture. (A) DNase I effect on biofilm formation, (B) DNase I effect on 48-h biofilm, (C) Proteinase K effect on biofilm formation, (D) Proteinase K effect on 48-h biofilm. The colored columns refer to the enzyme concentrations (0.125, 0.25, 0.5, and 1 mg/ml). The y-axis represents absorbance at 570 nm. The bars represent the means with standard deviations for 3–5 samples.
    Figure Legend Snippet: Effect of DNase I and proteinase K on the biofilm formation (time zero) and on 48-h-old biofilm. Fusobacterium nucleatum type strain ATCC 25586 (F.n) and Porphyromonas gingivalis type strain ATCC 33277 (P.g 33277) bacterial species were tested when they were grown as monoculture or as dual species culture. (A) DNase I effect on biofilm formation, (B) DNase I effect on 48-h biofilm, (C) Proteinase K effect on biofilm formation, (D) Proteinase K effect on 48-h biofilm. The colored columns refer to the enzyme concentrations (0.125, 0.25, 0.5, and 1 mg/ml). The y-axis represents absorbance at 570 nm. The bars represent the means with standard deviations for 3–5 samples.

    Techniques Used:

    Comparison of (A) carbohydrate and (B) eDNA yields from the biofilm matrix samples treated with proteinase K enzyme and non-treated samples. The matrix of 5-day-old biofilm was treated with 5 µg/ml proteinase K at 37°C for 1 h. F.n, Fusobacterium nucleatum ATCC 2558; P.g W50, Porphyromonas gingivalis W50; P.g 381, P. gingivalis FDC381; P.g 33277, P. gingivalis ATCC 33277. The bars represent the means with standard deviations from five samples (carbohydrates) and three replicates (eDNA).
    Figure Legend Snippet: Comparison of (A) carbohydrate and (B) eDNA yields from the biofilm matrix samples treated with proteinase K enzyme and non-treated samples. The matrix of 5-day-old biofilm was treated with 5 µg/ml proteinase K at 37°C for 1 h. F.n, Fusobacterium nucleatum ATCC 2558; P.g W50, Porphyromonas gingivalis W50; P.g 381, P. gingivalis FDC381; P.g 33277, P. gingivalis ATCC 33277. The bars represent the means with standard deviations from five samples (carbohydrates) and three replicates (eDNA).

    Techniques Used:

    Effect of DNase I and proteinase K enzymes at a concentration of 1 mg/ml on biofilm formation, (A) time zero and (B) 48-h-old biofilm. The graphs show the biomass (µm 3 /µm 2 ), maximum thickness (µm), and average thickness (µm) of one point z-stack confocal images analysed by COMSTAT software program. F.n, Fusobacterium nucleatum ATCC 25586; P.g 33277, Porphyromonas gingivalis ATCC 33277.
    Figure Legend Snippet: Effect of DNase I and proteinase K enzymes at a concentration of 1 mg/ml on biofilm formation, (A) time zero and (B) 48-h-old biofilm. The graphs show the biomass (µm 3 /µm 2 ), maximum thickness (µm), and average thickness (µm) of one point z-stack confocal images analysed by COMSTAT software program. F.n, Fusobacterium nucleatum ATCC 25586; P.g 33277, Porphyromonas gingivalis ATCC 33277.

    Techniques Used: Concentration Assay, Software

    5) Product Images from "Efficient Transmission and Characterization of Creutzfeldt-Jakob Disease Strains in Bank Voles"

    Article Title: Efficient Transmission and Characterization of Creutzfeldt-Jakob Disease Strains in Bank Voles

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.0020012

    Determination of the Molecular Type of PrP Sc Produced in Voles following Transmission of sCJD and gCJD (A) Immunoblot of proteinase K–resistant PrP Sc from sCJD and gCJD subtypes in human patients and after first passage in voles. PrP Sc produced in voles after primary transmission of mouse-passaged MM1 sCJD and MV1 sCJD, and of the mouse-adapted scrapie strain ME7 is also shown. The identity of the brain sample is designated above each lane. (B) Comparison of proteinase K–resistant PrP Sc produced in voles following first and second passages of MV1 sCJD, MM2 sCJD, E200K gCJD, and V210I gCJD. PrP Sc produced in voles after first passage of the mouse-adapted scrapie strain ME7 is also shown. The identity of the brain sample is designated above each lane. (C) Scatter-graph of proportions of di-glycosylated and mono-glycosylated PrP Sc in human patients (denoted by filled circles, filled inverted triangles, filled diamonds, filled squares, and filled upright triangles) and voles (denoted by open circles, open inverted triangles, open diamonds, open squares, open upright triangles, and asterisks), with MM1 sCJD (denoted by filled squares and open squares), MV1 sCJD (denoted by filled diamonds and open diamonds), MM2 sCJD (denoted by filled circles and open circles), V210I gCJD (denoted by filled inverted triangles and open inverted triangles), E200K gCJD (denoted by filled upright triangles and open upright triangles), and ME7 (denoted by asterisks). (D) Comparison of proteinase K–resistant PrP Sc produced in voles following inoculation with MM1 sCJD, MM2 sCJD, and the mouse-adapted scrapie strain ME7. Strep-tagged molecular markers (25 kDa and 20 kDa) are shown. The identity of the brain samples is designated above each lane.
    Figure Legend Snippet: Determination of the Molecular Type of PrP Sc Produced in Voles following Transmission of sCJD and gCJD (A) Immunoblot of proteinase K–resistant PrP Sc from sCJD and gCJD subtypes in human patients and after first passage in voles. PrP Sc produced in voles after primary transmission of mouse-passaged MM1 sCJD and MV1 sCJD, and of the mouse-adapted scrapie strain ME7 is also shown. The identity of the brain sample is designated above each lane. (B) Comparison of proteinase K–resistant PrP Sc produced in voles following first and second passages of MV1 sCJD, MM2 sCJD, E200K gCJD, and V210I gCJD. PrP Sc produced in voles after first passage of the mouse-adapted scrapie strain ME7 is also shown. The identity of the brain sample is designated above each lane. (C) Scatter-graph of proportions of di-glycosylated and mono-glycosylated PrP Sc in human patients (denoted by filled circles, filled inverted triangles, filled diamonds, filled squares, and filled upright triangles) and voles (denoted by open circles, open inverted triangles, open diamonds, open squares, open upright triangles, and asterisks), with MM1 sCJD (denoted by filled squares and open squares), MV1 sCJD (denoted by filled diamonds and open diamonds), MM2 sCJD (denoted by filled circles and open circles), V210I gCJD (denoted by filled inverted triangles and open inverted triangles), E200K gCJD (denoted by filled upright triangles and open upright triangles), and ME7 (denoted by asterisks). (D) Comparison of proteinase K–resistant PrP Sc produced in voles following inoculation with MM1 sCJD, MM2 sCJD, and the mouse-adapted scrapie strain ME7. Strep-tagged molecular markers (25 kDa and 20 kDa) are shown. The identity of the brain samples is designated above each lane.

    Techniques Used: Produced, Transmission Assay

    6) Product Images from ""

    Article Title:

    Journal:

    doi: 10.1074/jbc.M109.086587

    Insertion of GrB into a proteinase K-resistant mitochondrial compartment. A and B , mitochondrial localization of GrB. HeLa cells stably transfected with vector control or Bcl-2 were treated with GrB/Ad (33 n m /10 pfu/ml, respectively) for 10 min. The cells
    Figure Legend Snippet: Insertion of GrB into a proteinase K-resistant mitochondrial compartment. A and B , mitochondrial localization of GrB. HeLa cells stably transfected with vector control or Bcl-2 were treated with GrB/Ad (33 n m /10 pfu/ml, respectively) for 10 min. The cells

    Techniques Used: Stable Transfection, Transfection, Plasmid Preparation

    Subcellular localization of GrB Hax-1 cleavage products. A , translocation of in vitro translated full-length Hax-1 and the N-terminal Hax-1 fragment, but not the C-terminal Hax-1 fragment, into a proteinase K-resistant compartment of purified mitochondria.
    Figure Legend Snippet: Subcellular localization of GrB Hax-1 cleavage products. A , translocation of in vitro translated full-length Hax-1 and the N-terminal Hax-1 fragment, but not the C-terminal Hax-1 fragment, into a proteinase K-resistant compartment of purified mitochondria.

    Techniques Used: Translocation Assay, In Vitro, Purification

    7) Product Images from "Reoxidation of the Thiol-Disulfide Oxidoreductase MdbA by a Bacterial Vitamin K Epoxide Reductase in the Biofilm-Forming Actinobacterium Actinomyces oris"

    Article Title: Reoxidation of the Thiol-Disulfide Oxidoreductase MdbA by a Bacterial Vitamin K Epoxide Reductase in the Biofilm-Forming Actinobacterium Actinomyces oris

    Journal:

    doi: 10.1128/JB.00817-16

    Membrane topology of A. oris VKOR. (A) Presented is a topological model of the membrane protein VKOR, which possesses 6 cysteine (C) residues. C175 and C178 are part of the catalytic CXXC motif. To detect VKOR, a recombinant VKOR protein harboring a C-terminal double HA tag (2HA) was generated; in addition, the N-terminal 56-amino-acid epitope was used to raise polyclonal antibodies (square bracket). (B) Cell fractionation was performed on the parental strain MG1 and its isogenic ΔVKOR deletion mutants containing an empty vector (EV) or the complementing plasmid pVKOR-2HA. Protein samples isolated from culture medium (S), cell wall (W), membrane (M), and cytoplasmic (C) fractions were blotted with antibodies against the first 56 amino acids of VKOR (α-VKOR) and the 2×HA tag (α-HA). Anti-MdbA Ab (α-MdbA) was used to detect the membrane-bound MdbA. (C to F) Ultrathin sections of cells of the indicated strains were subjected to immunoelectron microscopy, for which samples were treated with anti-HA (C and E) or anti-VKOR (D and F) Ab, followed by IgG-conjugated gold particles (12 nm). The samples were viewed using an electron microscope after staining with 1% uranyl acetate. Scale bars indicate 100 nm. (G) Protoplasts of the ΔVKOR mutant expressing HA-tagged VKOR (pVKOR-2HA) were treated with increasing concentrations of proteinase K. The protein samples obtained were immunoblotted with anti-VKOR and anti-HA Ab. Molecular mass markers are indicated.
    Figure Legend Snippet: Membrane topology of A. oris VKOR. (A) Presented is a topological model of the membrane protein VKOR, which possesses 6 cysteine (C) residues. C175 and C178 are part of the catalytic CXXC motif. To detect VKOR, a recombinant VKOR protein harboring a C-terminal double HA tag (2HA) was generated; in addition, the N-terminal 56-amino-acid epitope was used to raise polyclonal antibodies (square bracket). (B) Cell fractionation was performed on the parental strain MG1 and its isogenic ΔVKOR deletion mutants containing an empty vector (EV) or the complementing plasmid pVKOR-2HA. Protein samples isolated from culture medium (S), cell wall (W), membrane (M), and cytoplasmic (C) fractions were blotted with antibodies against the first 56 amino acids of VKOR (α-VKOR) and the 2×HA tag (α-HA). Anti-MdbA Ab (α-MdbA) was used to detect the membrane-bound MdbA. (C to F) Ultrathin sections of cells of the indicated strains were subjected to immunoelectron microscopy, for which samples were treated with anti-HA (C and E) or anti-VKOR (D and F) Ab, followed by IgG-conjugated gold particles (12 nm). The samples were viewed using an electron microscope after staining with 1% uranyl acetate. Scale bars indicate 100 nm. (G) Protoplasts of the ΔVKOR mutant expressing HA-tagged VKOR (pVKOR-2HA) were treated with increasing concentrations of proteinase K. The protein samples obtained were immunoblotted with anti-VKOR and anti-HA Ab. Molecular mass markers are indicated.

    Techniques Used: Recombinant, Hemagglutination Assay, Generated, Cell Fractionation, Plasmid Preparation, Isolation, Immuno-Electron Microscopy, Microscopy, Staining, Mutagenesis, Expressing

    8) Product Images from "Transmembrane but not soluble helices fold inside the ribosome tunnel"

    Article Title: Transmembrane but not soluble helices fold inside the ribosome tunnel

    Journal: Nature Communications

    doi: 10.1038/s41467-018-07554-7

    Folding depends on hydrophobic length and correlates with insertion. a In vitro translation in the absence (−) and presence (+) of dog pancreas rough microsomes (RM) of truncated mRNAs of the same length (distance P-NST 67) harboring VSV-G full length (lanes 1 and 2) or half (VSV-G TM.5, lanes 3 and 4) TM segment, or gp41 full length (lanes 5 and 6) or half (gp41 TM.5, lanes 7 and 8) TM segment. Glycosylated and non-glycosylated molecules are indicated by black and white dots, respectively. Amino acid sequences included are shown on top. b Schematic of the engineered leader peptidase (Lep) model protein. Lep, consisting of 2 TM segments (H1 and H2) and a large luminal domain (P2), inserts into RMs in an N lum -C lum orientation. In vitro protein translation in the presence (+) or absence (–) of rough microsomes (RM) and proteinase K (PK) of VSV-G ( c ) or gp41 ( d ) derived sequences. Non-glycosylated protein bands are indicated by a white dot; single and double glycosylated protein bands are indicated by one or two black dots, respectively. An upwards black triangle indicates small protected singly glycosylated H2/inserted fragment. A double downward black triangle indicated large doubly glycosylated H2/G1/trasnlocated/G2/P2 fragment. Source data are provided as a Source Data file
    Figure Legend Snippet: Folding depends on hydrophobic length and correlates with insertion. a In vitro translation in the absence (−) and presence (+) of dog pancreas rough microsomes (RM) of truncated mRNAs of the same length (distance P-NST 67) harboring VSV-G full length (lanes 1 and 2) or half (VSV-G TM.5, lanes 3 and 4) TM segment, or gp41 full length (lanes 5 and 6) or half (gp41 TM.5, lanes 7 and 8) TM segment. Glycosylated and non-glycosylated molecules are indicated by black and white dots, respectively. Amino acid sequences included are shown on top. b Schematic of the engineered leader peptidase (Lep) model protein. Lep, consisting of 2 TM segments (H1 and H2) and a large luminal domain (P2), inserts into RMs in an N lum -C lum orientation. In vitro protein translation in the presence (+) or absence (–) of rough microsomes (RM) and proteinase K (PK) of VSV-G ( c ) or gp41 ( d ) derived sequences. Non-glycosylated protein bands are indicated by a white dot; single and double glycosylated protein bands are indicated by one or two black dots, respectively. An upwards black triangle indicates small protected singly glycosylated H2/inserted fragment. A double downward black triangle indicated large doubly glycosylated H2/G1/trasnlocated/G2/P2 fragment. Source data are provided as a Source Data file

    Techniques Used: In Vitro, Derivative Assay

    9) Product Images from "The TRiC Chaperonin Controls Reovirus Replication through Outer-Capsid Folding"

    Article Title: The TRiC Chaperonin Controls Reovirus Replication through Outer-Capsid Folding

    Journal: Nature microbiology

    doi: 10.1038/s41564-018-0122-x

    The TRiC chaperonin folds σ3 into a native, assembly-competent conformation a , Schematic of σ3 folding and assembly experiments. b , SDS-PAGE of 35 S-met-labeled σ3 immunoprecipitated from RRLs using a conformation-specific antibody (10C1 monoclonal antibody) or isotype control. Where indicated, RRLs were TRiC-depleted and reconstituted with purified hTRiC (+, 0.25 μM) (10% input loaded into lanes 1,4,7). c , SDS-PAGE of 35 S-met-labeled σ3 translated in RRLs mock-depleted, TRiC-depleted, or TRiC-depleted and reconstituted with purified hTRiC (+, 0.25 μM) and incubated with proteinase K (2.5 μg/mL final concentration) for the times shown. d , SDS-PAGE and colloidal blue stain of T1L reovirus virions and ISVPs. e , SDS-PAGE of ISVPs recoated with 35 S-met-labeled σ3 translated in RRLs and immunoprecipitated using a μ1δ-specific antibody, which is specific to the μ1 species on ISVPs. Where indicated, RRLs were TRiC-depleted and reconstituted with purified hTRiC (+, 0.125 μM; ++, 0.50 μM) (20% input loaded into lanes 1,4,7,10). Arrows in SDS-PAGE gels indicate full-length σ3. In b–e , three independent experiments were conducted with similar results. f , Model of the TRiC-σ3 folding and assembly pathway.
    Figure Legend Snippet: The TRiC chaperonin folds σ3 into a native, assembly-competent conformation a , Schematic of σ3 folding and assembly experiments. b , SDS-PAGE of 35 S-met-labeled σ3 immunoprecipitated from RRLs using a conformation-specific antibody (10C1 monoclonal antibody) or isotype control. Where indicated, RRLs were TRiC-depleted and reconstituted with purified hTRiC (+, 0.25 μM) (10% input loaded into lanes 1,4,7). c , SDS-PAGE of 35 S-met-labeled σ3 translated in RRLs mock-depleted, TRiC-depleted, or TRiC-depleted and reconstituted with purified hTRiC (+, 0.25 μM) and incubated with proteinase K (2.5 μg/mL final concentration) for the times shown. d , SDS-PAGE and colloidal blue stain of T1L reovirus virions and ISVPs. e , SDS-PAGE of ISVPs recoated with 35 S-met-labeled σ3 translated in RRLs and immunoprecipitated using a μ1δ-specific antibody, which is specific to the μ1 species on ISVPs. Where indicated, RRLs were TRiC-depleted and reconstituted with purified hTRiC (+, 0.125 μM; ++, 0.50 μM) (20% input loaded into lanes 1,4,7,10). Arrows in SDS-PAGE gels indicate full-length σ3. In b–e , three independent experiments were conducted with similar results. f , Model of the TRiC-σ3 folding and assembly pathway.

    Techniques Used: SDS Page, Labeling, Immunoprecipitation, Purification, Incubation, Concentration Assay, Staining

    10) Product Images from "A fluorescent imaging method for analyzing the biodistribution of therapeutic monoclonal antibodies that can distinguish intact antibodies from their breakdown products"

    Article Title: A fluorescent imaging method for analyzing the biodistribution of therapeutic monoclonal antibodies that can distinguish intact antibodies from their breakdown products

    Journal:

    doi: 10.1080/19420862.2015.1038683

    Unmixing analyses of the mixture of labeled trastuzumab and its products broken down by proteinase K treatment. ( A ) The unmixed images of the mixture of labeled intact trastuzumab and the breakdown products. Six mixtures with predefined ratios of breakdown
    Figure Legend Snippet: Unmixing analyses of the mixture of labeled trastuzumab and its products broken down by proteinase K treatment. ( A ) The unmixed images of the mixture of labeled intact trastuzumab and the breakdown products. Six mixtures with predefined ratios of breakdown

    Techniques Used: Labeling

    Antibodies labeled with 2 species of fluorophores. ( A ) The model of FRET-type labeled antibody. ( B ) Fluorescent spectra of intact XenoLight680 750-trastuzumab and the breakdown products digested by proteinase K treatment. The solutions were excited
    Figure Legend Snippet: Antibodies labeled with 2 species of fluorophores. ( A ) The model of FRET-type labeled antibody. ( B ) Fluorescent spectra of intact XenoLight680 750-trastuzumab and the breakdown products digested by proteinase K treatment. The solutions were excited

    Techniques Used: Labeling

    11) Product Images from "Folding–function relationship of the most common cystic fibrosis–causing CFTR conductance mutants"

    Article Title: Folding–function relationship of the most common cystic fibrosis–causing CFTR conductance mutants

    Journal: Life Science Alliance

    doi: 10.26508/lsa.201800172

    The proline in R347P destabilizes TM6. (A) As in Fig 3B , HEK293 cells transiently expressing indicated CFTR constructs were labeled for 15 min and chased for 0 or 2 h. The cells were lysed in 1% Triton X-100 in MNT and incubated with proteinase K for 15 min, followed by immunoprecipitation of CFTR with domain-specific antibodies. The lane labeled C is a nontransfected control. * indicates nonspecific bands. (B) Lane profiles of IP:TMD1 data shown in (A). (C) Helical wheel representation of CFTR residues 331–360, showing that R347 is largely surrounded by small hydrophobic residues. Hydrophilic (circle), hydrophobic (diamond), negatively charged (triangle), positively charged (pentagons). Shades of green indicate the hydrophobicity with dark green having high hydrophobicity and yellow zero hydrophobicity. Hydrophilic residues are shades of red, more intense red means most hydrophilic residue. Potentially charged residues are light blue. (D) Cryo-EM structure of human CFTR illustrating R347 and interacting residues, helices are shown as cylinders ( Liu et al, 2017 ) (PBD: 5UAK ). R347 is highlighted in red, and its interacting residues D993 and D924 are colored yellow. Transmembrane helix 6 and the N terminus (AA 1–77) are colored in light blue. Extracellular loops and intracellular loop 1 (ICL1) are labeled in dark gray. The colors wheat, blue, gray, green, and purple indicate TMD1, NBD1, R region, TMD2, and NBD2, respectively. The arrow indicates rotation in relation to Fig 3A left panel. (E) Same as in D, R117 in ECL1 and R334 in ECL2 are shown as red spheres, and their interacting residues E1126 in ECL5 and E217 in ECL3 are represented as yellow spheres. (F) Structural model of CFTR in the open conformation ( Mornon et al, 2015 ), illustrating R117 and R334 in red along with their binding partners in yellow. Colors and annotations are the same as in (D, E). Source data are available online for this figure.
    Figure Legend Snippet: The proline in R347P destabilizes TM6. (A) As in Fig 3B , HEK293 cells transiently expressing indicated CFTR constructs were labeled for 15 min and chased for 0 or 2 h. The cells were lysed in 1% Triton X-100 in MNT and incubated with proteinase K for 15 min, followed by immunoprecipitation of CFTR with domain-specific antibodies. The lane labeled C is a nontransfected control. * indicates nonspecific bands. (B) Lane profiles of IP:TMD1 data shown in (A). (C) Helical wheel representation of CFTR residues 331–360, showing that R347 is largely surrounded by small hydrophobic residues. Hydrophilic (circle), hydrophobic (diamond), negatively charged (triangle), positively charged (pentagons). Shades of green indicate the hydrophobicity with dark green having high hydrophobicity and yellow zero hydrophobicity. Hydrophilic residues are shades of red, more intense red means most hydrophilic residue. Potentially charged residues are light blue. (D) Cryo-EM structure of human CFTR illustrating R347 and interacting residues, helices are shown as cylinders ( Liu et al, 2017 ) (PBD: 5UAK ). R347 is highlighted in red, and its interacting residues D993 and D924 are colored yellow. Transmembrane helix 6 and the N terminus (AA 1–77) are colored in light blue. Extracellular loops and intracellular loop 1 (ICL1) are labeled in dark gray. The colors wheat, blue, gray, green, and purple indicate TMD1, NBD1, R region, TMD2, and NBD2, respectively. The arrow indicates rotation in relation to Fig 3A left panel. (E) Same as in D, R117 in ECL1 and R334 in ECL2 are shown as red spheres, and their interacting residues E1126 in ECL5 and E217 in ECL3 are represented as yellow spheres. (F) Structural model of CFTR in the open conformation ( Mornon et al, 2015 ), illustrating R117 and R334 in red along with their binding partners in yellow. Colors and annotations are the same as in (D, E). Source data are available online for this figure.

    Techniques Used: Expressing, Construct, Labeling, Incubation, Immunoprecipitation, Electron Microscopy, Binding Assay

    VX-809 corrects R347P folding. (A) HEK293 cells expressing CFTR constructs were labeled for 15 min and chased for 0 or 2 h in the presence or absence of VX-809. The cells were lysed in 1% Triton X-100 in MNT, and the cell lysates were incubated with or without Proteinase K at 25 μg/ml for 15 min. CFTR and fragments were immunoprecipitated using domain-specific antibodies as in Fig 3 . (B) Cell-surface biotinylation of CFTR in HEK293 cells with or without VX-809 or VX-770 pretreatment. The cells were lysed in 1% Triton X-100 in MNT, and the lysates were used for pull-down of biotinylated proteins with NeutrAvidin beads. 7.5% SDS-PAA gels were run and transferred to PVDF membrane and blotted against CFTR (596) or Actin. (C) Same as in (A) but in the presence of VX-770, VX-809, or both as indicated. All lanes of IP:TMD1 were present on one gel, but the solid black line indicates where the lanes were removed. * indicates nonspecific bands. Source data are available online for this figure.
    Figure Legend Snippet: VX-809 corrects R347P folding. (A) HEK293 cells expressing CFTR constructs were labeled for 15 min and chased for 0 or 2 h in the presence or absence of VX-809. The cells were lysed in 1% Triton X-100 in MNT, and the cell lysates were incubated with or without Proteinase K at 25 μg/ml for 15 min. CFTR and fragments were immunoprecipitated using domain-specific antibodies as in Fig 3 . (B) Cell-surface biotinylation of CFTR in HEK293 cells with or without VX-809 or VX-770 pretreatment. The cells were lysed in 1% Triton X-100 in MNT, and the lysates were used for pull-down of biotinylated proteins with NeutrAvidin beads. 7.5% SDS-PAA gels were run and transferred to PVDF membrane and blotted against CFTR (596) or Actin. (C) Same as in (A) but in the presence of VX-770, VX-809, or both as indicated. All lanes of IP:TMD1 were present on one gel, but the solid black line indicates where the lanes were removed. * indicates nonspecific bands. Source data are available online for this figure.

    Techniques Used: Expressing, Construct, Labeling, Incubation, Immunoprecipitation

    VX-770 does not improve R347P folding. (A) Cryo-EM structure of human CFTR ( Liu et al, 2017 ) (PBD: 5UAK ), generated using Chimera software ( Pettersen et al, 2004 ), illustrating that R117 is in the cytoplasm in the first extracellular loop between TM1 and TM2, R334 is located in the second extracellular loop between TM3 and TM4, and R347 is located in TM6 inside the pore. The colors wheat, purple, gray, green, and blue indicate TMD1, NBD1, R region, TMD2, and NBD2, respectively. (B) CFTR was expressed in HEK293 cells, which were labeled with 35 S-methionine/cysteine for 15 min and chased for 0 and 2 h in the presence or absence of 3 μM VX-770. The cells were lysed in 1% Triton X-100 in MNT, and the cell lysates were treated or not with Proteinase K at 25 μg/ml for 15 min. CFTR and fragments were immunoprecipitated using TMD1C (TMD1), Mr. Pink (NBD1 and full-length CFTR), TMD2C (TMD2), and 596 (NBD2) antibodies. * indicates nonspecific bands. Source data are available online for this figure.
    Figure Legend Snippet: VX-770 does not improve R347P folding. (A) Cryo-EM structure of human CFTR ( Liu et al, 2017 ) (PBD: 5UAK ), generated using Chimera software ( Pettersen et al, 2004 ), illustrating that R117 is in the cytoplasm in the first extracellular loop between TM1 and TM2, R334 is located in the second extracellular loop between TM3 and TM4, and R347 is located in TM6 inside the pore. The colors wheat, purple, gray, green, and blue indicate TMD1, NBD1, R region, TMD2, and NBD2, respectively. (B) CFTR was expressed in HEK293 cells, which were labeled with 35 S-methionine/cysteine for 15 min and chased for 0 and 2 h in the presence or absence of 3 μM VX-770. The cells were lysed in 1% Triton X-100 in MNT, and the cell lysates were treated or not with Proteinase K at 25 μg/ml for 15 min. CFTR and fragments were immunoprecipitated using TMD1C (TMD1), Mr. Pink (NBD1 and full-length CFTR), TMD2C (TMD2), and 596 (NBD2) antibodies. * indicates nonspecific bands. Source data are available online for this figure.

    Techniques Used: Electron Microscopy, Generated, Software, Labeling, Immunoprecipitation

    12) Product Images from "Non-consensus heptamer sequences destabilize the RAG post-cleavage complex, making ends available to alternative DNA repair pathways"

    Article Title: Non-consensus heptamer sequences destabilize the RAG post-cleavage complex, making ends available to alternative DNA repair pathways

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp1252

    Certain non-consensus RSS affect handling of SEs after cleavage. ( A ) Homologous recombination assay. Repair of a non-functional CFP gene by homologous recombination after RAG cleavage of plasmid 289-ntCFP leads to the expression of the CFP gene resulting in the detection of blue fluorescent cells, measurable by FACS analysis. ( B ) Quantification of FACS data ( n = 6). Background, as measured by CFP expression in cells transfected with a catalytically inactive RAG1 allele, DDE, was subtracted from each sample. Con denotes consensus RSS. ( C ) Southern blot showing products of RAG cleavage of 289-ntCFP by cores RAG1 and RAG2. Hirt preps were digested with HpaI and NcoI to generate a 1626 bp fragment. Cleavage by the V(D)J recombinase generates a pair of SEs that are 816 bp (12RSS) and 558 bp (23RSS) and can be visualized using an internally radiolabeled probe. First four lanes show a titration of Hirt DNA from a transfection with a consensus pair of RSS. Percent of DNA loaded is found above each lane. Lanes 4–10 show levels of SEs in vivo at various non-consensus RSSs, labeled above each lane. ( D ) Quantification of in vitro cleavage by purified RAG proteins, measured as the combined amount of radioactivity from cleaved products divided by the total amount of radioactivity. Examples of gels are shown in Figure 1 C in the proteinase K treated lane. * denotes P
    Figure Legend Snippet: Certain non-consensus RSS affect handling of SEs after cleavage. ( A ) Homologous recombination assay. Repair of a non-functional CFP gene by homologous recombination after RAG cleavage of plasmid 289-ntCFP leads to the expression of the CFP gene resulting in the detection of blue fluorescent cells, measurable by FACS analysis. ( B ) Quantification of FACS data ( n = 6). Background, as measured by CFP expression in cells transfected with a catalytically inactive RAG1 allele, DDE, was subtracted from each sample. Con denotes consensus RSS. ( C ) Southern blot showing products of RAG cleavage of 289-ntCFP by cores RAG1 and RAG2. Hirt preps were digested with HpaI and NcoI to generate a 1626 bp fragment. Cleavage by the V(D)J recombinase generates a pair of SEs that are 816 bp (12RSS) and 558 bp (23RSS) and can be visualized using an internally radiolabeled probe. First four lanes show a titration of Hirt DNA from a transfection with a consensus pair of RSS. Percent of DNA loaded is found above each lane. Lanes 4–10 show levels of SEs in vivo at various non-consensus RSSs, labeled above each lane. ( D ) Quantification of in vitro cleavage by purified RAG proteins, measured as the combined amount of radioactivity from cleaved products divided by the total amount of radioactivity. Examples of gels are shown in Figure 1 C in the proteinase K treated lane. * denotes P

    Techniques Used: Homologous Recombination, Functional Assay, Plasmid Preparation, Expressing, FACS, Transfection, Southern Blot, Titration, In Vivo, Labeling, In Vitro, Purification, Radioactivity

    Alterations to the RSS sequence can destabilize the RAG SE complex. ( A ) Schematic diagram of a RSS and its adjacent coding sequence. The consensus nucleotide sequence is shown. ( B ) Biochemical end release assay. Purified GST-tagged core RAG proteins cleave a ∼500 bp PCR product at 37°C. Post-cleavage SE complexes are thermally challenged at increasing temperatures and released SEs detected by electrophoresis. ( C ) Representative gels for end release assays. UR, unrearranged substrate; SC, single cleavage; CE, coding ends; SE, signal ends. Numbers above each lane indicate the temperatures reactions were heated to before electrophoresis. Samples treated with proteinase K and SDS are indicated with +pk. ( D ) Quantification of SE release, measured as the combined amount of radioactivity from cleaved products divided by the total amount of radioactivity, from three experiments using two different protein preparations (* P
    Figure Legend Snippet: Alterations to the RSS sequence can destabilize the RAG SE complex. ( A ) Schematic diagram of a RSS and its adjacent coding sequence. The consensus nucleotide sequence is shown. ( B ) Biochemical end release assay. Purified GST-tagged core RAG proteins cleave a ∼500 bp PCR product at 37°C. Post-cleavage SE complexes are thermally challenged at increasing temperatures and released SEs detected by electrophoresis. ( C ) Representative gels for end release assays. UR, unrearranged substrate; SC, single cleavage; CE, coding ends; SE, signal ends. Numbers above each lane indicate the temperatures reactions were heated to before electrophoresis. Samples treated with proteinase K and SDS are indicated with +pk. ( D ) Quantification of SE release, measured as the combined amount of radioactivity from cleaved products divided by the total amount of radioactivity, from three experiments using two different protein preparations (* P

    Techniques Used: Sequencing, Release Assay, Purification, Polymerase Chain Reaction, Electrophoresis, Radioactivity

    13) Product Images from "Evolutionary Regression and Species-Specific Codon Usage of TLR15"

    Article Title: Evolutionary Regression and Species-Specific Codon Usage of TLR15

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.02626

    NF-κB activation by reptilian TLR15. HEK293 cells transiently transfected with an NF-κB luciferase reporter plasmid and empty vector, chicken (gaga), anolis (anca), crocodile (cpro), alligator (almi) TLR15 or the gagaTLR2B and gagaTLR1A plasmids were stimulated (5 h) with Proteinase K (100 ng/mL), Pam 3 CSK 4 (100 ng/mL), FSL-1 (100 ng/mL), 10 μL of Chrysosporium anamorph of Nannizziopsis vriesii (CANV) sterile culture supernatant or 10 μL of CANV supernatant pre-treated (30 min) with 1 mM PMSF. Values are the mean ± SEM fold increase of NF-κB activity, represented by luciferase activity in Relative Light Units (RLU), in stimulated cells over unstimulated control cells from three independent experiments performed in duplicate.
    Figure Legend Snippet: NF-κB activation by reptilian TLR15. HEK293 cells transiently transfected with an NF-κB luciferase reporter plasmid and empty vector, chicken (gaga), anolis (anca), crocodile (cpro), alligator (almi) TLR15 or the gagaTLR2B and gagaTLR1A plasmids were stimulated (5 h) with Proteinase K (100 ng/mL), Pam 3 CSK 4 (100 ng/mL), FSL-1 (100 ng/mL), 10 μL of Chrysosporium anamorph of Nannizziopsis vriesii (CANV) sterile culture supernatant or 10 μL of CANV supernatant pre-treated (30 min) with 1 mM PMSF. Values are the mean ± SEM fold increase of NF-κB activity, represented by luciferase activity in Relative Light Units (RLU), in stimulated cells over unstimulated control cells from three independent experiments performed in duplicate.

    Techniques Used: Activation Assay, Transfection, Luciferase, Plasmid Preparation, Activity Assay

    Proteolytic cleavage and expression of reptilian TLR15. (A) Immunoblot analysis of HEK293 cells expressing C-terminally FLAG-tagged chicken (gaga), anolis (anca), crocodile (cpro), or alligator (almi) TLR15 left untreated (–) or stimulated (+) (1 h) with 250 ng/mL Proteinase K. Mature TLR15 is ~140 kDa. Treatment with Proteinase K results in cleavage of gagaTLR15 and ancaTLR15 to form a cleaved receptor fragment that is slightly higher than 70 kDa. Note that crpoTLR15 and almiTLR15 are poorly expressed compared to ancaTLR15 and gagaTLR15. Beta-actin was detected to confirm equal loading of total protein onto SDS-PAGE gel. (B) Confocal microscopy on HEK293 cells expressing C-terminally HA-tagged TLR15 (green). Note that crpoTLR15 and almiTLR15 show lower expression compared to ancaTLR15 and gagaTLR15. All images were produced with the same microscopy settings. Nuclei are stained with DAPI (blue). White scale bar is 10 μm. Three representative images from two independent experiments are shown for each transfected group. (C) Immunoblot analysis of reptilian viper heart (VH-2) cells transfected with the different FLAG-tagged TLR15s. The rabbit α-human Beta actin antibody cross reacts with a specific protein in VH-2 cell lysate which was used to confirm equal loading of total protein onto SDS-PAGE gel. For (A,C) ; results are representative of three independent experiments.
    Figure Legend Snippet: Proteolytic cleavage and expression of reptilian TLR15. (A) Immunoblot analysis of HEK293 cells expressing C-terminally FLAG-tagged chicken (gaga), anolis (anca), crocodile (cpro), or alligator (almi) TLR15 left untreated (–) or stimulated (+) (1 h) with 250 ng/mL Proteinase K. Mature TLR15 is ~140 kDa. Treatment with Proteinase K results in cleavage of gagaTLR15 and ancaTLR15 to form a cleaved receptor fragment that is slightly higher than 70 kDa. Note that crpoTLR15 and almiTLR15 are poorly expressed compared to ancaTLR15 and gagaTLR15. Beta-actin was detected to confirm equal loading of total protein onto SDS-PAGE gel. (B) Confocal microscopy on HEK293 cells expressing C-terminally HA-tagged TLR15 (green). Note that crpoTLR15 and almiTLR15 show lower expression compared to ancaTLR15 and gagaTLR15. All images were produced with the same microscopy settings. Nuclei are stained with DAPI (blue). White scale bar is 10 μm. Three representative images from two independent experiments are shown for each transfected group. (C) Immunoblot analysis of reptilian viper heart (VH-2) cells transfected with the different FLAG-tagged TLR15s. The rabbit α-human Beta actin antibody cross reacts with a specific protein in VH-2 cell lysate which was used to confirm equal loading of total protein onto SDS-PAGE gel. For (A,C) ; results are representative of three independent experiments.

    Techniques Used: Expressing, SDS Page, Confocal Microscopy, Hemagglutination Assay, Produced, Microscopy, Staining, Transfection

    14) Product Images from "Chaperone-mediated coupling of endoplasmic reticulum and mitochondrial Ca2+ channels"

    Article Title: Chaperone-mediated coupling of endoplasmic reticulum and mitochondrial Ca2+ channels

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200608073

    IP 3 R, VDAC1, and grp75 colocalize on the MAM fraction. (A) Protein components of subcellular fractions prepared from rat liver and HeLa cells revealed by immunoblot analysis. Mito, mitochondria; MAM, light mitochondrial fraction; P, heavy mitochondrial fraction, enriched in matrix components; C, crude mitochondrial fraction before Percoll gradient separation. 10 μg of proteins were loaded on 10% SDS-polyacrylamide gels. The presence of IP 3 Rs was shown by using a non–isotype-specific monoclonal antibody. VDAC1 and grp75 were both present in the MAM, whereas it was free of contamination from inner membrane (Cox-II) and matrix (MnSOD; C) proteins. Different preparations are separated by the dotted line. Blots are representative of more than five experiments. (B) Blue-native and SDS-PAGE 2D separation of the MAM fraction (below BN) and Mito P proteins (above BN; for preparation of native subcellular fractions, see Materials and methods and A). The native fractions were solubilized and separated on an acrylamide gradient gel in the first dimension. The capillary gel was stacked over a 10% SDS-polyacrylamide gel and separated, and the proteins were immunoblotted against the IP 3 Rs, grp75, and VDAC1. A typical result of an immunoblot from three separate experiments is shown. (C) The MAM and Mito P fractions (50 μg of proteins) were subjected to proteinase K digestion (50 μg/ml) and the presence of grp75 and MnSOD was revealed by immunoblotting. Hyposmotic shock (50 mM mannitol, 5 mM Hepes, and 0.1 mM EGTA for 30 min at room temperature) was applied to the Mito P fraction to induce release of matrix proteins. (D–F) Coimmunoprecipitation of grp75 with IP 3 R and VDAC1. Total cellular proteins were used for immunoprecipitation with a polyclonal IP 3 R1 (D), a polyclonal VDAC (E), and a monoclonal grp75 (F) antibody, and the precipitated protein fractions were separated on 10% SDS-polyacrylamide gels and immunoblotted against IP 3 Rs, grp75, and VDAC1. The input homogenate fractions, the IgG controls, and the immunoprecipitates are shown.
    Figure Legend Snippet: IP 3 R, VDAC1, and grp75 colocalize on the MAM fraction. (A) Protein components of subcellular fractions prepared from rat liver and HeLa cells revealed by immunoblot analysis. Mito, mitochondria; MAM, light mitochondrial fraction; P, heavy mitochondrial fraction, enriched in matrix components; C, crude mitochondrial fraction before Percoll gradient separation. 10 μg of proteins were loaded on 10% SDS-polyacrylamide gels. The presence of IP 3 Rs was shown by using a non–isotype-specific monoclonal antibody. VDAC1 and grp75 were both present in the MAM, whereas it was free of contamination from inner membrane (Cox-II) and matrix (MnSOD; C) proteins. Different preparations are separated by the dotted line. Blots are representative of more than five experiments. (B) Blue-native and SDS-PAGE 2D separation of the MAM fraction (below BN) and Mito P proteins (above BN; for preparation of native subcellular fractions, see Materials and methods and A). The native fractions were solubilized and separated on an acrylamide gradient gel in the first dimension. The capillary gel was stacked over a 10% SDS-polyacrylamide gel and separated, and the proteins were immunoblotted against the IP 3 Rs, grp75, and VDAC1. A typical result of an immunoblot from three separate experiments is shown. (C) The MAM and Mito P fractions (50 μg of proteins) were subjected to proteinase K digestion (50 μg/ml) and the presence of grp75 and MnSOD was revealed by immunoblotting. Hyposmotic shock (50 mM mannitol, 5 mM Hepes, and 0.1 mM EGTA for 30 min at room temperature) was applied to the Mito P fraction to induce release of matrix proteins. (D–F) Coimmunoprecipitation of grp75 with IP 3 R and VDAC1. Total cellular proteins were used for immunoprecipitation with a polyclonal IP 3 R1 (D), a polyclonal VDAC (E), and a monoclonal grp75 (F) antibody, and the precipitated protein fractions were separated on 10% SDS-polyacrylamide gels and immunoblotted against IP 3 Rs, grp75, and VDAC1. The input homogenate fractions, the IgG controls, and the immunoprecipitates are shown.

    Techniques Used: SDS Page, Immunoprecipitation

    15) Product Images from "Site-specific "

    Article Title: Site-specific

    Journal: Molecular & Cellular Proteomics : MCP

    doi: 10.1074/mcp.M115.053546

    Fragment ion spectra of the Proteinase K generated plasminogen O -glycopeptide 362 LAP T APPELTPV 373 measured with nanoRP-LC-ESI MS n (positive ion mode, CID and ETD). A (top), For the given O -glycopeptide the CID-MS 2 spectrum is shown together with its corresponding precursor ion m / z 718.30 [M+3H] 3+ (inset). The spectrum allows the elucidation of the O -glycan composition (here disialylated T-antigen). In addition, also some internal glycopeptide fragments have been detected ( e.g. b 10 +HexNAc). A (bottom): The putative peptide mass ( m / z 1205.66 [M+H] + ) of the given O -glycopeptide was subjected to CID-MS 3 fragmentation. The peptide was identified by MASCOT search (Score: 16, UniProt KB/Swiss-Prot, human). B, The O -glycosylation site (here Thr 365 ) was pinpointed by means of ETD (Biotools-Score: 150). Magnified regions show the isotope pattern of selected peptide fragment ions, confirming the annotation. In addition to peptide fragment ions also fragment ions derived from the glycan moiety were detected, allowing a verification of the glycan composition. Furthermore, a neutral loss of an acetyl radical from the intact O -glycopeptide was observed, which is typically seen in ETD spectra of glycopeptides.
    Figure Legend Snippet: Fragment ion spectra of the Proteinase K generated plasminogen O -glycopeptide 362 LAP T APPELTPV 373 measured with nanoRP-LC-ESI MS n (positive ion mode, CID and ETD). A (top), For the given O -glycopeptide the CID-MS 2 spectrum is shown together with its corresponding precursor ion m / z 718.30 [M+3H] 3+ (inset). The spectrum allows the elucidation of the O -glycan composition (here disialylated T-antigen). In addition, also some internal glycopeptide fragments have been detected ( e.g. b 10 +HexNAc). A (bottom): The putative peptide mass ( m / z 1205.66 [M+H] + ) of the given O -glycopeptide was subjected to CID-MS 3 fragmentation. The peptide was identified by MASCOT search (Score: 16, UniProt KB/Swiss-Prot, human). B, The O -glycosylation site (here Thr 365 ) was pinpointed by means of ETD (Biotools-Score: 150). Magnified regions show the isotope pattern of selected peptide fragment ions, confirming the annotation. In addition to peptide fragment ions also fragment ions derived from the glycan moiety were detected, allowing a verification of the glycan composition. Furthermore, a neutral loss of an acetyl radical from the intact O -glycopeptide was observed, which is typically seen in ETD spectra of glycopeptides.

    Techniques Used: Generated, Liquid Chromatography, Mass Spectrometry, Derivative Assay

    16) Product Images from "Site-specific Relaxase Activity of a VirD2-like Protein Encoded within the tfs4 Genomic Island of Helicobacter pylor"

    Article Title: Site-specific Relaxase Activity of a VirD2-like Protein Encoded within the tfs4 Genomic Island of Helicobacter pylor

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.496430

    Purification and DNA binding activity of Tfs4 VirD2. A , DNA co-fractionating in the MBP-VirD2 size exclusion chromatography fraction ( lane 1 ) is efficiently removed in a subsequent ion exchange step ( lane 2 ). B , three-stage purification of MBP-VirD2. *, purified MBP-Tfs4 VirD2 ( lane 1 ) and MBP-Tfs4 VirD2(N) ( lane 2 ) resolved by 10% SDS-PAGE. C , MBP-VirD2 DNA binding activity. Incubation of MBP-VirD2 with 100 ng of pSB14 (containing cloned RP4 oriT ) ( i ), pRD205 (pGEM-TEasy containing cloned tfs4 virD2 and upstream intergenic sequence) ( ii ), or pRD200 (pGEM-TEasy containing cloned tfs4 virD2 only) ( iii ) results in a decrease in supercoiled plasmid and concomitant increase in both open circle (nicked) forms and loading well-retarded nucleoprotein complexes ( black arrow ) as protein concentration increases. Effects are most pronounced with plasmid pRD205. Notably, linear plasmid is also absent from the pRD205 sample following proteinase K treatment. Lanes 1–6 , 0, 0.05, 0.1, 0.15, 0.3, and 0.5 pmol of MBP-VirD2; lane 7 , 0.5 pmol of MBP-VirD2 treated with proteinase K; lane 8 , linear plasmid generated by restriction enzyme digest. All reactions were performed at 37 °C in the presence of MgCl 2 . OC , open circle (nicked); SC , supercoiled, L , linear.
    Figure Legend Snippet: Purification and DNA binding activity of Tfs4 VirD2. A , DNA co-fractionating in the MBP-VirD2 size exclusion chromatography fraction ( lane 1 ) is efficiently removed in a subsequent ion exchange step ( lane 2 ). B , three-stage purification of MBP-VirD2. *, purified MBP-Tfs4 VirD2 ( lane 1 ) and MBP-Tfs4 VirD2(N) ( lane 2 ) resolved by 10% SDS-PAGE. C , MBP-VirD2 DNA binding activity. Incubation of MBP-VirD2 with 100 ng of pSB14 (containing cloned RP4 oriT ) ( i ), pRD205 (pGEM-TEasy containing cloned tfs4 virD2 and upstream intergenic sequence) ( ii ), or pRD200 (pGEM-TEasy containing cloned tfs4 virD2 only) ( iii ) results in a decrease in supercoiled plasmid and concomitant increase in both open circle (nicked) forms and loading well-retarded nucleoprotein complexes ( black arrow ) as protein concentration increases. Effects are most pronounced with plasmid pRD205. Notably, linear plasmid is also absent from the pRD205 sample following proteinase K treatment. Lanes 1–6 , 0, 0.05, 0.1, 0.15, 0.3, and 0.5 pmol of MBP-VirD2; lane 7 , 0.5 pmol of MBP-VirD2 treated with proteinase K; lane 8 , linear plasmid generated by restriction enzyme digest. All reactions were performed at 37 °C in the presence of MgCl 2 . OC , open circle (nicked); SC , supercoiled, L , linear.

    Techniques Used: Purification, Binding Assay, Activity Assay, Size-exclusion Chromatography, SDS Page, Incubation, Clone Assay, Sequencing, Plasmid Preparation, Protein Concentration, Generated

    Site-specific cleavage of oligonucleotides by Tfs4 VirD2. The indicated 5′ (Tfs4) or 3′ (Tfs4, Tfs4 mutated (mut), and RP4) DIG-labeled 30-mer oligonucleotides were incubated with 5 pmol of Tfs4 MBP-VirD2(N) and then subsequently in the presence or absence of either Proteinase K ( K ) or trypsin ( T ). The resulting oligonucleotide products and nucleoprotein-peptide complexes were resolved in denaturing 20% polyacrylamide gels and analyzed by Southern blotting. MBP-VirD2(N) cleaves the 3′-end of the 5′-DIG-labeled Tfs4 oligonucleotide (putative oriT ATCCTG-containing sequence upstream of virD2 in the tfs4 cluster) ( blot 1 ). The equivalent 3′-DIG-labeled oligonucleotide is retained in the gel well in the presence of MBP-VirD2(N) ( blot 2 ). Following protease treatment, cleaved ATCCTG-containing oligonucleotides demonstrate retarded gel migration due to the attachment of proteolyzed VirD2 peptides (D2 Tryp and D2 ProtK , blots 2 and 4 ). Cleavage and VirD2 peptide attachment to 3′-DIG-labeled oligonucleotides can be effectively abrogated by the addition of a 100-fold excess of competing unlabeled Tfs4 oligonucleotide ( C ) but not by the addition of non-competing random sequence oligonucleotide lacking the ATCCTG sequence ( N ) ( blot 2 ). Cleavage is similarly not observed following incubation of MBP-VirD2(N) with a Tfs4 3′-DIG-labeled oligonucleotide in which the ATCCTG sequence is entirely mutated ( mut ; blot 3 ). All reactions required the presence of MgCl 2 . Full oligonucleotide sequences are listed in Table 1 .
    Figure Legend Snippet: Site-specific cleavage of oligonucleotides by Tfs4 VirD2. The indicated 5′ (Tfs4) or 3′ (Tfs4, Tfs4 mutated (mut), and RP4) DIG-labeled 30-mer oligonucleotides were incubated with 5 pmol of Tfs4 MBP-VirD2(N) and then subsequently in the presence or absence of either Proteinase K ( K ) or trypsin ( T ). The resulting oligonucleotide products and nucleoprotein-peptide complexes were resolved in denaturing 20% polyacrylamide gels and analyzed by Southern blotting. MBP-VirD2(N) cleaves the 3′-end of the 5′-DIG-labeled Tfs4 oligonucleotide (putative oriT ATCCTG-containing sequence upstream of virD2 in the tfs4 cluster) ( blot 1 ). The equivalent 3′-DIG-labeled oligonucleotide is retained in the gel well in the presence of MBP-VirD2(N) ( blot 2 ). Following protease treatment, cleaved ATCCTG-containing oligonucleotides demonstrate retarded gel migration due to the attachment of proteolyzed VirD2 peptides (D2 Tryp and D2 ProtK , blots 2 and 4 ). Cleavage and VirD2 peptide attachment to 3′-DIG-labeled oligonucleotides can be effectively abrogated by the addition of a 100-fold excess of competing unlabeled Tfs4 oligonucleotide ( C ) but not by the addition of non-competing random sequence oligonucleotide lacking the ATCCTG sequence ( N ) ( blot 2 ). Cleavage is similarly not observed following incubation of MBP-VirD2(N) with a Tfs4 3′-DIG-labeled oligonucleotide in which the ATCCTG sequence is entirely mutated ( mut ; blot 3 ). All reactions required the presence of MgCl 2 . Full oligonucleotide sequences are listed in Table 1 .

    Techniques Used: Labeling, Incubation, Southern Blot, Sequencing, Migration

    17) Product Images from "Glycoprotein D Receptor-Dependent, Low-pH-Independent Endocytic Entry of Herpes Simplex Virus Type 1"

    Article Title: Glycoprotein D Receptor-Dependent, Low-pH-Independent Endocytic Entry of Herpes Simplex Virus Type 1

    Journal:

    doi: 10.1128/JVI.79.11.6655-6663.2005

    Rapid, gD receptor-dependent protection of viral glycoproteins from proteinase K digestion. A. Virus was attached to B78 and C10 cells for 45 min at 4°C. Entry was then initiated by incubation at 37°C for the indicated times. Cells were
    Figure Legend Snippet: Rapid, gD receptor-dependent protection of viral glycoproteins from proteinase K digestion. A. Virus was attached to B78 and C10 cells for 45 min at 4°C. Entry was then initiated by incubation at 37°C for the indicated times. Cells were

    Techniques Used: Incubation

    Protection of viral gB from proteinase K correlates with endocytic entry. After virus attachment for 45 min at 4°C, cells were either held at 4°C (lanes 1 and 3) or incubated at 37°C for 10 min (lanes 2 and 4) and then chilled
    Figure Legend Snippet: Protection of viral gB from proteinase K correlates with endocytic entry. After virus attachment for 45 min at 4°C, cells were either held at 4°C (lanes 1 and 3) or incubated at 37°C for 10 min (lanes 2 and 4) and then chilled

    Techniques Used: Incubation

    18) Product Images from "Novel Type of Chronic Wasting Disease Detected in Moose (Alces alces), Norway"

    Article Title: Novel Type of Chronic Wasting Disease Detected in Moose (Alces alces), Norway

    Journal: Emerging Infectious Diseases

    doi: 10.3201/eid2412.180702

    Comparison of protease-resistant PrP res from moose ( Alces alces ) with chronic wasting disease and from sheep with scrapie, Europe. Representative blots show epitope mapping analysis of PrP res (lane 4, CH1641; lane 5, moose no. 1; lane 6, moose no. 2) in comparison with different ovine transmissible spongiform encephalopathy isolates (lane 1, atypical/Nor98; lane 2, classical scrapie; and lane 3, CH1641). A chronic wasting disease isolate from Canada was loaded as control (lane 7). The antibodies used are indicated on the left. Protein standards are shown in lane M (10, 15, 20, 25, 37, and 50 kDa). The small amount of PrP res with intact 12B2 epitope in moose no.1 had a molecular weight higher than that observed with more C-terminal monoclonal antibodies (18.7 + 0.3 kDa measured with 12B2 vs. 17.2 + 0.1 kDa measured with L42). Even if the increase of the apparent molecular weight might be a known behavior when proteinase K cleavage occurs near the epitope, we noted that, in the case of moose no. 1, the 12B2-positive PrP res had a molecular weight higher than scrapie (18.1 + 0.1 kDa measured with 12B2) and CH1641-like sample (18.1 + 0.4 kDa when detected with 12B2). PrP res , protease-resistant core of abnormal form of prion protein.
    Figure Legend Snippet: Comparison of protease-resistant PrP res from moose ( Alces alces ) with chronic wasting disease and from sheep with scrapie, Europe. Representative blots show epitope mapping analysis of PrP res (lane 4, CH1641; lane 5, moose no. 1; lane 6, moose no. 2) in comparison with different ovine transmissible spongiform encephalopathy isolates (lane 1, atypical/Nor98; lane 2, classical scrapie; and lane 3, CH1641). A chronic wasting disease isolate from Canada was loaded as control (lane 7). The antibodies used are indicated on the left. Protein standards are shown in lane M (10, 15, 20, 25, 37, and 50 kDa). The small amount of PrP res with intact 12B2 epitope in moose no.1 had a molecular weight higher than that observed with more C-terminal monoclonal antibodies (18.7 + 0.3 kDa measured with 12B2 vs. 17.2 + 0.1 kDa measured with L42). Even if the increase of the apparent molecular weight might be a known behavior when proteinase K cleavage occurs near the epitope, we noted that, in the case of moose no. 1, the 12B2-positive PrP res had a molecular weight higher than scrapie (18.1 + 0.1 kDa measured with 12B2) and CH1641-like sample (18.1 + 0.4 kDa when detected with 12B2). PrP res , protease-resistant core of abnormal form of prion protein.

    Techniques Used: Molecular Weight

    19) Product Images from "Phenotyping of circulating extracellular vesicles (EVs) in obesity identifies large EVs as functional conveyors of Macrophage Migration Inhibitory Factor"

    Article Title: Phenotyping of circulating extracellular vesicles (EVs) in obesity identifies large EVs as functional conveyors of Macrophage Migration Inhibitory Factor

    Journal: Molecular Metabolism

    doi: 10.1016/j.molmet.2018.10.001

    MIF specifically uses MVs as a secretory pathway . A . MIF concentration significantly decreased upon removal of plasma MVs. MIF concentration, successively measured in PFP, PFP w/o MVs, and PFP w/o EVs is presented in control patients (left, n  = 19), overweight patients (middle, n  = 19), and obese patients (right, n  = 19). B . MV-associated MIF ratios are unchanged with obesity. Percent of EV-associated fractions and soluble MIF fraction was calculated based on MIF concentrations measured in PFP, PFP w/o MV, and PFP w/o EV. MVs carry around half of circulating MIF in control, overweight and obese patients. C–D . Western blots of EV preparations confirmed specific MIF association with MVs. MV and EXO preparations isolated from plasma patients (C) or from 3T3-L1 adipocytes and T-CEM lymphocytes supernatants (D) were analyzed for MIF presence. MIF specifically associated with MVs, whatever EV cellular origin. β-actin and CD9 were respectively used as MV and EXO protein markers. E . MIF is located within MVs. Proteinase K treatment did not modify MV-associated MIF signal by comparison to untreated MVs excluding that MIF is exposed on MVs. Combined treatment of proteinase K and Triton X100 led to MIF signal disappearance, establishing that MIF is packed within MVs. The transmembrane protein CD9 and the cytoplasmic spindle-oriented caveolin are respectively used as positive controls of this proteinase K protection assay.
    Figure Legend Snippet: MIF specifically uses MVs as a secretory pathway . A . MIF concentration significantly decreased upon removal of plasma MVs. MIF concentration, successively measured in PFP, PFP w/o MVs, and PFP w/o EVs is presented in control patients (left, n  = 19), overweight patients (middle, n  = 19), and obese patients (right, n  = 19). B . MV-associated MIF ratios are unchanged with obesity. Percent of EV-associated fractions and soluble MIF fraction was calculated based on MIF concentrations measured in PFP, PFP w/o MV, and PFP w/o EV. MVs carry around half of circulating MIF in control, overweight and obese patients. C–D . Western blots of EV preparations confirmed specific MIF association with MVs. MV and EXO preparations isolated from plasma patients (C) or from 3T3-L1 adipocytes and T-CEM lymphocytes supernatants (D) were analyzed for MIF presence. MIF specifically associated with MVs, whatever EV cellular origin. β-actin and CD9 were respectively used as MV and EXO protein markers. E . MIF is located within MVs. Proteinase K treatment did not modify MV-associated MIF signal by comparison to untreated MVs excluding that MIF is exposed on MVs. Combined treatment of proteinase K and Triton X100 led to MIF signal disappearance, establishing that MIF is packed within MVs. The transmembrane protein CD9 and the cytoplasmic spindle-oriented caveolin are respectively used as positive controls of this proteinase K protection assay.

    Techniques Used: Concentration Assay, Western Blot, Isolation

    20) Product Images from "Nature and Lability of Northern Adriatic Macroaggregates"

    Article Title: Nature and Lability of Northern Adriatic Macroaggregates

    Journal: Marine Drugs

    doi: 10.3390/md8092480

    FT-IR spectra of the surface macroaggregate matrix and aqueous phase of experimental slurries after α-amylase + β-glucosidase and protease + proteinase K hydrolysis.
    Figure Legend Snippet: FT-IR spectra of the surface macroaggregate matrix and aqueous phase of experimental slurries after α-amylase + β-glucosidase and protease + proteinase K hydrolysis.

    Techniques Used:

    FT-IR spectra of the ( A ) surface and ( B ) water column macroaggregate matrices, and after (i) α-amylase + β-glucosidase, (ii) protease + proteinase K hydrolysis: carbohydrate bands (region ~1150–900 cm −1 ), protein bands (region 1654–1635 cm −1 ), lipid bands (region 2950–2850 cm −1 ) and inorganic (mineral) components (region
    Figure Legend Snippet: FT-IR spectra of the ( A ) surface and ( B ) water column macroaggregate matrices, and after (i) α-amylase + β-glucosidase, (ii) protease + proteinase K hydrolysis: carbohydrate bands (region ~1150–900 cm −1 ), protein bands (region 1654–1635 cm −1 ), lipid bands (region 2950–2850 cm −1 ) and inorganic (mineral) components (region

    Techniques Used:

    Related Articles

    Clone Assay:

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    Article Snippet: CDNAs encoding MCAD, NDUFA9 and COX VIa-L (liver isoform) were cloned into the pGEM4Z vector (Promega, Madison, WI, USA) and proteins translated using the TnT Coupled Reticulocyte Lysate System (Promega) in the presence of [35 S]-methionine/cysteine. .. Samples subjected to protease treatment were incubated on ice for 10 min with 100 μg/mL proteinase K (Sigma) before treatment with 1 mM PMSF for 10 min.

    Centrifugation:

    Article Title: The PPE Domain of PPE17 Is Responsible for Its Surface Localization and Can Be Used to Express Heterologous Proteins on the Mycobacterial Surface
    Article Snippet: The assay was performed as previously described with some modifications: cells were grown to an OD600 of about 0.8, harvested by centrifugation at 3000×g for 10 minutes at room temperature, washed twice in TBST buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM MgCl2 and 0.05% Tween 80). .. One aliquot was treated with proteinase K (Sigma-Aldrich) 100 µg ml−1 , whereas the other was left untreated and incubated for 30 min at 4°C.

    Article Title: Tyrosine phosphatase SHP2 negatively regulates NLRP3 inflammasome activation via ANT1-dependent mitochondrial homeostasis
    Article Snippet: For protease digestion, fractions of mitochondria (resuspended in 20 mM HEPES-KOH, pH 7.4, 250 mM sucrose, 80 mM KOAc, and 5 mM MgOAc) were incubated with 40 μM proteinase K (Sigma-Aldrich) for 30 min on ice. .. For protease digestion, fractions of mitochondria (resuspended in 20 mM HEPES-KOH, pH 7.4, 250 mM sucrose, 80 mM KOAc, and 5 mM MgOAc) were incubated with 40 μM proteinase K (Sigma-Aldrich) for 30 min on ice.

    Article Title: Characterization of the pgf operon involved in the posttranslational modification of Streptococcus mutans surface proteins
    Article Snippet: Susceptibility of Cnm and WapA to protease degradation was determined as previously described . .. Briefly, cells from overnight cultures of the indicated S . mutans strains were pelleted by centrifugation and resuspended in 1 × PBS pH 7.2 containing increasing amounts of proteinase K (Sigma-Aldrich). .. After 30 min incubation on ice, protease activity was neutralized by addition of a protease inhibitor cocktail for 5 min (Thermo Scientific).

    Amplification:

    Article Title: Non-consensus heptamer sequences destabilize the RAG post-cleavage complex, making ends available to alternative DNA repair pathways
    Article Snippet: Reactions were then aliquoted into microfuge tubes and incubated at different temperatures, as indicated, or treated with stop buffer [10 mM Tris (pH 8.0), 10 mM EDTA, 0.2% SDS, 0.35 mg/ml proteinase K (Sigma Aldrich)] for 30 min and then ran out on 4–20% acrylamide TBE gels (Invitrogen). .. Reactions were then aliquoted into microfuge tubes and incubated at different temperatures, as indicated, or treated with stop buffer [10 mM Tris (pH 8.0), 10 mM EDTA, 0.2% SDS, 0.35 mg/ml proteinase K (Sigma Aldrich)] for 30 min and then ran out on 4–20% acrylamide TBE gels (Invitrogen).

    Zymography:

    Article Title: Dendritic Cell-derived Extracellular Vesicles mediate Mesenchymal Stem/Stromal Cell recruitment
    Article Snippet: Paragraph title: Gelatin zymography ... Where indicated, EV and 100 K supernatant samples were incubated with proteinase K (10 μg/mL, 15 min, 37 °C, Sigma-Aldrich), for soluble proteins digestion, before or after EV lysis.

    Construct:

    Article Title: Rapid experimental SAD phasing and hot-spot identification with halogenated fragments
    Article Snippet: Prior to crystallization, the HIV-1 RT construct RT52A (20 mg ml−1 ; Bauman, Patel, Dharia et al. , 2013 ) was incubated with rilpivirine (TMC278/Edurant) at a 1:1.5 protein:drug molar ratio at room temperature (∼23°C) for 30 min. RT–rilpivirine crystals were produced in hanging drops at 4°C with a 1:1 ratio of protein solution and well solution consisting of 11%(v /v ) PEG 8000, 4%(v /v ) PEG 400, 50 mM imidazole pH 6.6, 10 mM spermine, 15 mM MgSO4 , 100 mM ammonium sulfate, 5 mM tris(2-carboxyethyl)phos­phine together with an experimentally optimized concentration of microseeds from previously generated and crushed RT–rilpivirine crystals (pre-seeding). .. Proteinase K was purchased from Sigma–Aldrich (St Louis, Missouri, USA) and crystallized as described previously (Beck et al. , 2010 ).

    Article Title: Non-consensus heptamer sequences destabilize the RAG post-cleavage complex, making ends available to alternative DNA repair pathways
    Article Snippet: Reactions were then aliquoted into microfuge tubes and incubated at different temperatures, as indicated, or treated with stop buffer [10 mM Tris (pH 8.0), 10 mM EDTA, 0.2% SDS, 0.35 mg/ml proteinase K (Sigma Aldrich)] for 30 min and then ran out on 4–20% acrylamide TBE gels (Invitrogen). .. DNA was visualized using SYBR safe DNA gel stain (Invitrogen).

    Enzyme-linked Immunosorbent Assay:

    Article Title: The PPE Domain of PPE17 Is Responsible for Its Surface Localization and Can Be Used to Express Heterologous Proteins on the Mycobacterial Surface
    Article Snippet: Paragraph title: Enzyme-linked Immunosorbent Assay with Whole Cells of M. smegmatis or M. bovis BCG ... One aliquot was treated with proteinase K (Sigma-Aldrich) 100 µg ml−1 , whereas the other was left untreated and incubated for 30 min at 4°C.

    Incubation:

    Article Title: A short purification process for quantitative isolation of PrPSc from naturally occurring and experimental transmissible spongiform encephalopathies
    Article Snippet: For limiting dilution experiments, the polytron was replaced with the OmniGLH homogenizer (CAMLAB, UK) which worked equally well with disposable probes, a very useful factor to be considered while attempting multiple homogenizations with large number of samples. .. On the day of use, 100 μl of homogenate was diluted with an equal volume of the homogenization buffer and incubated with proteinase K (Sigma-Aldrich) for 1 hour at 37°C with mild rocking. .. Concentrations of proteinase K used varied with the species and are provided separately with each figure.

    Article Title: Rapid experimental SAD phasing and hot-spot identification with halogenated fragments
    Article Snippet: Prior to crystallization, the HIV-1 RT construct RT52A (20 mg ml−1 ; Bauman, Patel, Dharia et al. , 2013 ) was incubated with rilpivirine (TMC278/Edurant) at a 1:1.5 protein:drug molar ratio at room temperature (∼23°C) for 30 min. RT–rilpivirine crystals were produced in hanging drops at 4°C with a 1:1 ratio of protein solution and well solution consisting of 11%(v /v ) PEG 8000, 4%(v /v ) PEG 400, 50 mM imidazole pH 6.6, 10 mM spermine, 15 mM MgSO4 , 100 mM ammonium sulfate, 5 mM tris(2-carboxyethyl)phos­phine together with an experimentally optimized concentration of microseeds from previously generated and crushed RT–rilpivirine crystals (pre-seeding). .. Proteinase K was purchased from Sigma–Aldrich (St Louis, Missouri, USA) and crystallized as described previously (Beck et al. , 2010 ).

    Article Title: The PPE Domain of PPE17 Is Responsible for Its Surface Localization and Can Be Used to Express Heterologous Proteins on the Mycobacterial Surface
    Article Snippet: Pellets were finally resuspended in 1 ml of TBST and divided into two identical aliquots. .. One aliquot was treated with proteinase K (Sigma-Aldrich) 100 µg ml−1 , whereas the other was left untreated and incubated for 30 min at 4°C. .. The reaction was stopped adding 1X complete EDTA-free protease inhibitor (Roche).

    Article Title: The PPE Domain of PPE17 Is Responsible for Its Surface Localization and Can Be Used to Express Heterologous Proteins on the Mycobacterial Surface
    Article Snippet: Each sample was divided in two identical aliquots. .. One aliquot was treated with proteinase K (Sigma-Aldrich) up to a concentration of100 µg ml−1 , whereas the other was left untreated and incubated for 30 min at 4°C. .. The reaction was stopped by the addition of 1X complete EDTA-free protease inhibitor (Roche).

    Article Title: Distinct SagA from Hospital-Associated Clade A1 Enterococcus faecium Strains Contributes to Biofilm Formation
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    Article Title: Loss of the Mitochondrial Fatty Acid β-Oxidation Protein Medium-Chain Acyl-Coenzyme A Dehydrogenase Disrupts Oxidative Phosphorylation Protein Complex Stability and Function
    Article Snippet: Dissipation of the mitochondrial membrane potential (Δψm ) was performed in the presence of 10 µM FCCP (with no ATP or sodium succinate). .. Samples subjected to protease treatment were incubated on ice for 10 min with 100 μg/mL proteinase K (Sigma) before treatment with 1 mM PMSF for 10 min. .. Protein band intensities were calculated using ImageJ (NIH) software from at least three independent experiments.

    Article Title: Tyrosine phosphatase SHP2 negatively regulates NLRP3 inflammasome activation via ANT1-dependent mitochondrial homeostasis
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    Article Title: Dendritic Cell-derived Extracellular Vesicles mediate Mesenchymal Stem/Stromal Cell recruitment
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    Article Title: Topology of the Maize Mixed Linkage (1- > 3),(1- > 4)-?-D-Glucan Synthase at the Golgi Membrane
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    Article Title: Cotranslational Folding Inhibits Translocation from Within the Ribosome–Sec61 Translocon Complex
    Article Snippet: All samples were analyzed by SDS-PAGE on 10–18% gradient gels and imaged on KODAK imaging screens using a BioRad personalFx phosphorimager and Quantity One software (BioRad, Hercules, CA). .. Translation reactions were incubated with 0.1 mg/ml proteinase K (EMD Millipore, Darmstadt, Germany) for 1 hr at 4°C. .. Protease digestion was stopped by addition of phenylmethylsulfonylfluoride (1 mM final concentration) followed by immediate immersion in 10 volumes of 98°C 1% SDS (wt/vol), 0.1M Tris-HCl, pH 6.8.

    Article Title: Stabilization of Urinary MicroRNAs by Association with Exosomes and Argonaute 2 Protein
    Article Snippet: Following analysis, the experiment was then repeated at the 30 min time-point using 10 samples. .. Urine samples from 2 control subjects were incubated at 55 °C ±50 μg/mL of proteinase K (P-2308; Sigma-Aldrich, Gillingham, Dorset, UK). .. Aliquots of 250 μL were removed after 0, 10, 20, 30, 40, 50 and 60 min to which 750 μL of QIAzol plus 1 μg of carrier RNA was added, and samples were then stored at −80 °C.

    Article Title: Molecular Analysis of Cases of Italian Sheep Scrapie and Comparison with Cases of Bovine Spongiform Encephalopathy (BSE) and Experimental BSE in Sheep
    Article Snippet: The homogenate was incubated for 20 min at room temperature and then centrifuged at 22,000 × g for 20 min (TLA 100.3 rotor; Beckman). .. Supernatant (1 ml) was added with 50 μl of a stock solution of proteinase K (Sigma) to give a final concentration of 50 μg/ml and then incubated for 1 h at 37°C with gentle shaking. .. Protease treatment was stopped with phenylmethylsulfonyl fluoride (PMSF) (Sigma) (30 μl of a stock solution, to give a final concentration of 3 mM).

    Article Title: Non-consensus heptamer sequences destabilize the RAG post-cleavage complex, making ends available to alternative DNA repair pathways
    Article Snippet: For RAG-mediated cleavage, 100 ng of recombination substrate (PCR product from 289-nt CFP) was incubated for 3 h at 37°C with 200 ng purified RAG protein and 200 ng of purified recombinant HMGB1 in a buffer containing 50 mM Hepes (pH 8.0), 25 mM KCl, 4 mM NaCl, 1 mM DTT, 0.1 μg BSA, 5 mM CaCl2 and 5 mM MgCl2 ( ). .. Reactions were then aliquoted into microfuge tubes and incubated at different temperatures, as indicated, or treated with stop buffer [10 mM Tris (pH 8.0), 10 mM EDTA, 0.2% SDS, 0.35 mg/ml proteinase K (Sigma Aldrich)] for 30 min and then ran out on 4–20% acrylamide TBE gels (Invitrogen). .. DNA was visualized using SYBR safe DNA gel stain (Invitrogen).

    Activity Assay:

    Article Title: Dendritic Cell-derived Extracellular Vesicles mediate Mesenchymal Stem/Stromal Cell recruitment
    Article Snippet: Gelatinase activity of MMPs was analysed on conditioned media collected separately from TC and BC compartments of transwell assay, and processed as described before , or directly on EV pellets and 100 K supernatants. .. Where indicated, EV and 100 K supernatant samples were incubated with proteinase K (10 μg/mL, 15 min, 37 °C, Sigma-Aldrich), for soluble proteins digestion, before or after EV lysis.

    Article Title: Topology of the Maize Mixed Linkage (1- > 3),(1- > 4)-?-D-Glucan Synthase at the Golgi Membrane
    Article Snippet: A CHAPS pretreatment was carried out at 4°C for 30 min. Proteinase K (EC 3.4.21.64, Sigma) in RB was then added to reach concentrations indicated in a final reaction volume of 1.5 mL, and the mixtures were incubated at 30°C for 30 min. .. A CHAPS pretreatment was carried out at 4°C for 30 min. Proteinase K (EC 3.4.21.64, Sigma) in RB was then added to reach concentrations indicated in a final reaction volume of 1.5 mL, and the mixtures were incubated at 30°C for 30 min.

    Mass Spectrometry:

    Article Title: The major secreted protein Msp1/p75 is O-glycosylated in Lactobacillus rhamnosus GG
    Article Snippet: Paragraph title: MS determinations ... The excised gel pieces containing Msp1 were reduced, alkylated and digested with trypsin (Promega), as previously described [ ] or with Proteinase K (from T. album ; Sigma-Aldrich) [ ].

    BIA-KA:

    Article Title: Identification of the active components in Bone Marrow Soup: a mitigator against irradiation-injury to salivary glands
    Article Snippet: The micro-centrifuge tube cap was wrapped with parafilm to avoid evaporation; 2) Heated at 95 °C for 60 min; 3) Digested by Trypsin (1:20 w/w, T1426, Sigma-Aldrich, ST. Louis, USA) overnight at 37 °C; and 4) Digested by 0.1 μg/μl proteinase K (P2308 Sigma-Aldrich, ST. Louis, USA) overnight at 37 °C. .. Precipitates were removed by micro-centrifugation by 13,500 rpm for 15 min. 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, 120V, 60 min) and Coomassie blue staining were conducted to test the efficiency of deactivation.

    Western Blot:

    Article Title: Characterization of the pgf operon involved in the posttranslational modification of Streptococcus mutans surface proteins
    Article Snippet: Briefly, cells from overnight cultures of the indicated S . mutans strains were pelleted by centrifugation and resuspended in 1 × PBS pH 7.2 containing increasing amounts of proteinase K (Sigma-Aldrich). .. After 30 min incubation on ice, protease activity was neutralized by addition of a protease inhibitor cocktail for 5 min (Thermo Scientific).

    Crystallization Assay:

    Article Title: Rapid experimental SAD phasing and hot-spot identification with halogenated fragments
    Article Snippet: Paragraph title: Crystallization   ... Proteinase K was purchased from Sigma–Aldrich (St Louis, Missouri, USA) and crystallized as described previously (Beck et al. , 2010 ).

    Produced:

    Article Title: Rapid experimental SAD phasing and hot-spot identification with halogenated fragments
    Article Snippet: Prior to crystallization, the HIV-1 RT construct RT52A (20 mg ml−1 ; Bauman, Patel, Dharia et al. , 2013 ) was incubated with rilpivirine (TMC278/Edurant) at a 1:1.5 protein:drug molar ratio at room temperature (∼23°C) for 30 min. RT–rilpivirine crystals were produced in hanging drops at 4°C with a 1:1 ratio of protein solution and well solution consisting of 11%(v /v ) PEG 8000, 4%(v /v ) PEG 400, 50 mM imidazole pH 6.6, 10 mM spermine, 15 mM MgSO4 , 100 mM ammonium sulfate, 5 mM tris(2-carboxyethyl)phos­phine together with an experimentally optimized concentration of microseeds from previously generated and crushed RT–rilpivirine crystals (pre-seeding). .. Proteinase K was purchased from Sigma–Aldrich (St Louis, Missouri, USA) and crystallized as described previously (Beck et al. , 2010 ).

    Flow Cytometry:

    Article Title: The major secreted protein Msp1/p75 is O-glycosylated in Lactobacillus rhamnosus GG
    Article Snippet: The excised gel pieces containing Msp1 were reduced, alkylated and digested with trypsin (Promega), as previously described [ ] or with Proteinase K (from T. album ; Sigma-Aldrich) [ ]. .. The excised gel pieces containing Msp1 were reduced, alkylated and digested with trypsin (Promega), as previously described [ ] or with Proteinase K (from T. album ; Sigma-Aldrich) [ ].

    Chromatography:

    Article Title: The major secreted protein Msp1/p75 is O-glycosylated in Lactobacillus rhamnosus GG
    Article Snippet: The LGG Msp1 protein purified by cation exchange chromatography was applied to SDS-PAGE using Nu-Page 4-12% gradient Bis-Tris (Invitrogen) gels. .. The excised gel pieces containing Msp1 were reduced, alkylated and digested with trypsin (Promega), as previously described [ ] or with Proteinase K (from T. album ; Sigma-Aldrich) [ ].

    Protease Inhibitor:

    Article Title: A short purification process for quantitative isolation of PrPSc from naturally occurring and experimental transmissible spongiform encephalopathies
    Article Snippet: On the day of use, 100 μl of homogenate was diluted with an equal volume of the homogenization buffer and incubated with proteinase K (Sigma-Aldrich) for 1 hour at 37°C with mild rocking. .. On the day of use, 100 μl of homogenate was diluted with an equal volume of the homogenization buffer and incubated with proteinase K (Sigma-Aldrich) for 1 hour at 37°C with mild rocking.

    Article Title: Stabilization of Urinary MicroRNAs by Association with Exosomes and Argonaute 2 Protein
    Article Snippet: Urine samples from 2 control subjects were incubated at 55 °C ±50 μg/mL of proteinase K (P-2308; Sigma-Aldrich, Gillingham, Dorset, UK). .. Urine samples from 2 control subjects were incubated at 55 °C ±50 μg/mL of proteinase K (P-2308; Sigma-Aldrich, Gillingham, Dorset, UK).

    Drug Susceptibility Assay:

    Article Title: Characterization of the pgf operon involved in the posttranslational modification of Streptococcus mutans surface proteins
    Article Snippet: Paragraph title: Proteinase K susceptibility assay ... Briefly, cells from overnight cultures of the indicated S . mutans strains were pelleted by centrifugation and resuspended in 1 × PBS pH 7.2 containing increasing amounts of proteinase K (Sigma-Aldrich).

    Generated:

    Article Title: Rapid experimental SAD phasing and hot-spot identification with halogenated fragments
    Article Snippet: Prior to crystallization, the HIV-1 RT construct RT52A (20 mg ml−1 ; Bauman, Patel, Dharia et al. , 2013 ) was incubated with rilpivirine (TMC278/Edurant) at a 1:1.5 protein:drug molar ratio at room temperature (∼23°C) for 30 min. RT–rilpivirine crystals were produced in hanging drops at 4°C with a 1:1 ratio of protein solution and well solution consisting of 11%(v /v ) PEG 8000, 4%(v /v ) PEG 400, 50 mM imidazole pH 6.6, 10 mM spermine, 15 mM MgSO4 , 100 mM ammonium sulfate, 5 mM tris(2-carboxyethyl)phos­phine together with an experimentally optimized concentration of microseeds from previously generated and crushed RT–rilpivirine crystals (pre-seeding). .. Proteinase K was purchased from Sigma–Aldrich (St Louis, Missouri, USA) and crystallized as described previously (Beck et al. , 2010 ).

    other:

    Article Title: Folding–function relationship of the most common cystic fibrosis–causing CFTR conductance mutants
    Article Snippet: In short, detergent cell lysates were digested for 15 min on ice, using 25 μg/ml proteinase K (Sigma-Aldrich).

    Article Title: Characterization of extracellular polymeric matrix, and treatment of Fusobacterium nucleatum and Porphyromonas gingivalis biofilms with DNase I and proteinase K
    Article Snippet: Deoxyribonuclease I (DNase I) (Sigma-Aldrich, MO, USA) from bovine pancreas was prepared in an enzyme buffer (0.15 mM NaCl and 5 mM MgCl2 ), and proteinase K (Sigma-Aldrich, MO, USA) was prepared in distilled water.

    Transwell Assay:

    Article Title: Dendritic Cell-derived Extracellular Vesicles mediate Mesenchymal Stem/Stromal Cell recruitment
    Article Snippet: Gelatinase activity of MMPs was analysed on conditioned media collected separately from TC and BC compartments of transwell assay, and processed as described before , or directly on EV pellets and 100 K supernatants. .. Where indicated, EV and 100 K supernatant samples were incubated with proteinase K (10 μg/mL, 15 min, 37 °C, Sigma-Aldrich), for soluble proteins digestion, before or after EV lysis.

    Enzyme Inhibition Assay:

    Article Title: Thiol reductive stress induces cellulose-anchored biofilm formation in Mycobacterium tuberculosis
    Article Snippet: Paragraph title: Enzyme inhibition assay ... Mtb biofilms were treated with cellulase (T. viride , Calbiochem) at 5 mg ml−1 in citrate buffer, 0.05 M, pH 4 α-amylase (672 U ml−1 ) from Bacillus licheniformis (Sigma), Turbo DNase (0.8 U ml−1 ) (Invitrogen), proteinase K (0.1 mg ml−1 , from Tritirachium album , Sigma) or lipase (0.5 mg ml−1 , from Chromobacterium viscosum , Calbiochem).

    Polymerase Chain Reaction:

    Article Title: Non-consensus heptamer sequences destabilize the RAG post-cleavage complex, making ends available to alternative DNA repair pathways
    Article Snippet: For RAG-mediated cleavage, 100 ng of recombination substrate (PCR product from 289-nt CFP) was incubated for 3 h at 37°C with 200 ng purified RAG protein and 200 ng of purified recombinant HMGB1 in a buffer containing 50 mM Hepes (pH 8.0), 25 mM KCl, 4 mM NaCl, 1 mM DTT, 0.1 μg BSA, 5 mM CaCl2 and 5 mM MgCl2 ( ). .. Reactions were then aliquoted into microfuge tubes and incubated at different temperatures, as indicated, or treated with stop buffer [10 mM Tris (pH 8.0), 10 mM EDTA, 0.2% SDS, 0.35 mg/ml proteinase K (Sigma Aldrich)] for 30 min and then ran out on 4–20% acrylamide TBE gels (Invitrogen).

    Injection:

    Article Title: The major secreted protein Msp1/p75 is O-glycosylated in Lactobacillus rhamnosus GG
    Article Snippet: The excised gel pieces containing Msp1 were reduced, alkylated and digested with trypsin (Promega), as previously described [ ] or with Proteinase K (from T. album ; Sigma-Aldrich) [ ]. .. The excised gel pieces containing Msp1 were reduced, alkylated and digested with trypsin (Promega), as previously described [ ] or with Proteinase K (from T. album ; Sigma-Aldrich) [ ].

    Recombinant:

    Article Title: Non-consensus heptamer sequences destabilize the RAG post-cleavage complex, making ends available to alternative DNA repair pathways
    Article Snippet: For RAG-mediated cleavage, 100 ng of recombination substrate (PCR product from 289-nt CFP) was incubated for 3 h at 37°C with 200 ng purified RAG protein and 200 ng of purified recombinant HMGB1 in a buffer containing 50 mM Hepes (pH 8.0), 25 mM KCl, 4 mM NaCl, 1 mM DTT, 0.1 μg BSA, 5 mM CaCl2 and 5 mM MgCl2 ( ). .. Reactions were then aliquoted into microfuge tubes and incubated at different temperatures, as indicated, or treated with stop buffer [10 mM Tris (pH 8.0), 10 mM EDTA, 0.2% SDS, 0.35 mg/ml proteinase K (Sigma Aldrich)] for 30 min and then ran out on 4–20% acrylamide TBE gels (Invitrogen).

    Gene Knockout:

    Article Title: Loss of the Mitochondrial Fatty Acid β-Oxidation Protein Medium-Chain Acyl-Coenzyme A Dehydrogenase Disrupts Oxidative Phosphorylation Protein Complex Stability and Function
    Article Snippet: Samples subjected to protease treatment were incubated on ice for 10 min with 100 μg/mL proteinase K (Sigma) before treatment with 1 mM PMSF for 10 min. .. Samples subjected to protease treatment were incubated on ice for 10 min with 100 μg/mL proteinase K (Sigma) before treatment with 1 mM PMSF for 10 min.

    Isolation:

    Article Title: Loss of the Mitochondrial Fatty Acid β-Oxidation Protein Medium-Chain Acyl-Coenzyme A Dehydrogenase Disrupts Oxidative Phosphorylation Protein Complex Stability and Function
    Article Snippet: Translation products were incubated with freshly isolated mitochondria in 250 mM sucrose, 80 mM potassium acetate, 5 mM magnesium acetate, 10 mM sodium succinate, 1 mM dithiothreitol, 5 mM ATP and 20 mM HEPES pH 7.4 at 37 °C for the times indicated. .. Samples subjected to protease treatment were incubated on ice for 10 min with 100 μg/mL proteinase K (Sigma) before treatment with 1 mM PMSF for 10 min.

    Article Title: Tyrosine phosphatase SHP2 negatively regulates NLRP3 inflammasome activation via ANT1-dependent mitochondrial homeostasis
    Article Snippet: The cytoplasmic and mitochondrial fractions were prepared by using the Mitochondria Isolation Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. .. For protease digestion, fractions of mitochondria (resuspended in 20 mM HEPES-KOH, pH 7.4, 250 mM sucrose, 80 mM KOAc, and 5 mM MgOAc) were incubated with 40 μM proteinase K (Sigma-Aldrich) for 30 min on ice.

    Article Title: Stabilization of Urinary MicroRNAs by Association with Exosomes and Argonaute 2 Protein
    Article Snippet: Urine samples from 2 control subjects were incubated at 55 °C ±50 μg/mL of proteinase K (P-2308; Sigma-Aldrich, Gillingham, Dorset, UK). .. Urine samples from 2 control subjects were incubated at 55 °C ±50 μg/mL of proteinase K (P-2308; Sigma-Aldrich, Gillingham, Dorset, UK).

    Protein Concentration:

    Article Title: Identification of the active components in Bone Marrow Soup: a mitigator against irradiation-injury to salivary glands
    Article Snippet: The micro-centrifuge tube cap was wrapped with parafilm to avoid evaporation; 2) Heated at 95 °C for 60 min; 3) Digested by Trypsin (1:20 w/w, T1426, Sigma-Aldrich, ST. Louis, USA) overnight at 37 °C; and 4) Digested by 0.1 μg/μl proteinase K (P2308 Sigma-Aldrich, ST. Louis, USA) overnight at 37 °C. .. Precipitates were removed by micro-centrifugation by 13,500 rpm for 15 min. 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, 120V, 60 min) and Coomassie blue staining were conducted to test the efficiency of deactivation.

    Size-exclusion Chromatography:

    Article Title: Non-consensus heptamer sequences destabilize the RAG post-cleavage complex, making ends available to alternative DNA repair pathways
    Article Snippet: Paragraph title: Thermal challenge assays to measure stability of the SEC ... Reactions were then aliquoted into microfuge tubes and incubated at different temperatures, as indicated, or treated with stop buffer [10 mM Tris (pH 8.0), 10 mM EDTA, 0.2% SDS, 0.35 mg/ml proteinase K (Sigma Aldrich)] for 30 min and then ran out on 4–20% acrylamide TBE gels (Invitrogen).

    Purification:

    Article Title: Rapid experimental SAD phasing and hot-spot identification with halogenated fragments
    Article Snippet: Influenza endonuclease was expressed, purified and crystallized as described previously (Bauman, Patel, Baker et al. , 2013 ). .. Proteinase K was purchased from Sigma–Aldrich (St Louis, Missouri, USA) and crystallized as described previously (Beck et al. , 2010 ).

    Article Title: The major secreted protein Msp1/p75 is O-glycosylated in Lactobacillus rhamnosus GG
    Article Snippet: The LGG Msp1 protein purified by cation exchange chromatography was applied to SDS-PAGE using Nu-Page 4-12% gradient Bis-Tris (Invitrogen) gels. .. The excised gel pieces containing Msp1 were reduced, alkylated and digested with trypsin (Promega), as previously described [ ] or with Proteinase K (from T. album ; Sigma-Aldrich) [ ].

    Article Title: Non-consensus heptamer sequences destabilize the RAG post-cleavage complex, making ends available to alternative DNA repair pathways
    Article Snippet: For RAG-mediated cleavage, 100 ng of recombination substrate (PCR product from 289-nt CFP) was incubated for 3 h at 37°C with 200 ng purified RAG protein and 200 ng of purified recombinant HMGB1 in a buffer containing 50 mM Hepes (pH 8.0), 25 mM KCl, 4 mM NaCl, 1 mM DTT, 0.1 μg BSA, 5 mM CaCl2 and 5 mM MgCl2 ( ). .. Reactions were then aliquoted into microfuge tubes and incubated at different temperatures, as indicated, or treated with stop buffer [10 mM Tris (pH 8.0), 10 mM EDTA, 0.2% SDS, 0.35 mg/ml proteinase K (Sigma Aldrich)] for 30 min and then ran out on 4–20% acrylamide TBE gels (Invitrogen).

    Degradation Assay:

    Article Title: The PPE Domain of PPE17 Is Responsible for Its Surface Localization and Can Be Used to Express Heterologous Proteins on the Mycobacterial Surface
    Article Snippet: Paragraph title: Proteinase K Degradation Assay ... One aliquot was treated with proteinase K (Sigma-Aldrich) up to a concentration of100 µg ml−1 , whereas the other was left untreated and incubated for 30 min at 4°C.

    Lysis:

    Article Title: The PPE Domain of PPE17 Is Responsible for Its Surface Localization and Can Be Used to Express Heterologous Proteins on the Mycobacterial Surface
    Article Snippet: One aliquot was treated with proteinase K (Sigma-Aldrich) up to a concentration of100 µg ml−1 , whereas the other was left untreated and incubated for 30 min at 4°C. .. Subsequently, samples were washed once in TBS and resuspended in TBS plus loading buffer 5X (sucrose 50% w/v, SDS 10% w/v, 0.3 M Tris HCl pH 6.8, bromophenol blue 0.05%w/v, β-mercaptoethanol 5% v/v) or subjected to subcellular fractionation as described below.

    Article Title: Dendritic Cell-derived Extracellular Vesicles mediate Mesenchymal Stem/Stromal Cell recruitment
    Article Snippet: Gelatinase activity of MMPs was analysed on conditioned media collected separately from TC and BC compartments of transwell assay, and processed as described before , or directly on EV pellets and 100 K supernatants. .. Where indicated, EV and 100 K supernatant samples were incubated with proteinase K (10 μg/mL, 15 min, 37 °C, Sigma-Aldrich), for soluble proteins digestion, before or after EV lysis. .. Protease was heat-inactivated (60 °C, 10 min) in the presence of phenylmethylsulfonyl fluoride 5 mM.

    Mouse Assay:

    Article Title: Identification of the active components in Bone Marrow Soup: a mitigator against irradiation-injury to salivary glands
    Article Snippet: The micro-centrifuge tube cap was wrapped with parafilm to avoid evaporation; 2) Heated at 95 °C for 60 min; 3) Digested by Trypsin (1:20 w/w, T1426, Sigma-Aldrich, ST. Louis, USA) overnight at 37 °C; and 4) Digested by 0.1 μg/μl proteinase K (P2308 Sigma-Aldrich, ST. Louis, USA) overnight at 37 °C. .. The micro-centrifuge tube cap was wrapped with parafilm to avoid evaporation; 2) Heated at 95 °C for 60 min; 3) Digested by Trypsin (1:20 w/w, T1426, Sigma-Aldrich, ST. Louis, USA) overnight at 37 °C; and 4) Digested by 0.1 μg/μl proteinase K (P2308 Sigma-Aldrich, ST. Louis, USA) overnight at 37 °C.

    SDS Page:

    Article Title: Tyrosine phosphatase SHP2 negatively regulates NLRP3 inflammasome activation via ANT1-dependent mitochondrial homeostasis
    Article Snippet: For protease digestion, fractions of mitochondria (resuspended in 20 mM HEPES-KOH, pH 7.4, 250 mM sucrose, 80 mM KOAc, and 5 mM MgOAc) were incubated with 40 μM proteinase K (Sigma-Aldrich) for 30 min on ice. .. For protease digestion, fractions of mitochondria (resuspended in 20 mM HEPES-KOH, pH 7.4, 250 mM sucrose, 80 mM KOAc, and 5 mM MgOAc) were incubated with 40 μM proteinase K (Sigma-Aldrich) for 30 min on ice.

    Article Title: The major secreted protein Msp1/p75 is O-glycosylated in Lactobacillus rhamnosus GG
    Article Snippet: The LGG Msp1 protein purified by cation exchange chromatography was applied to SDS-PAGE using Nu-Page 4-12% gradient Bis-Tris (Invitrogen) gels. .. The excised gel pieces containing Msp1 were reduced, alkylated and digested with trypsin (Promega), as previously described [ ] or with Proteinase K (from T. album ; Sigma-Aldrich) [ ].

    Plasmid Preparation:

    Article Title: Loss of the Mitochondrial Fatty Acid β-Oxidation Protein Medium-Chain Acyl-Coenzyme A Dehydrogenase Disrupts Oxidative Phosphorylation Protein Complex Stability and Function
    Article Snippet: CDNAs encoding MCAD, NDUFA9 and COX VIa-L (liver isoform) were cloned into the pGEM4Z vector (Promega, Madison, WI, USA) and proteins translated using the TnT Coupled Reticulocyte Lysate System (Promega) in the presence of [35 S]-methionine/cysteine. .. Samples subjected to protease treatment were incubated on ice for 10 min with 100 μg/mL proteinase K (Sigma) before treatment with 1 mM PMSF for 10 min.

    In Vitro:

    Article Title: Loss of the Mitochondrial Fatty Acid β-Oxidation Protein Medium-Chain Acyl-Coenzyme A Dehydrogenase Disrupts Oxidative Phosphorylation Protein Complex Stability and Function
    Article Snippet: Paragraph title: In vitro mitochondrial import assays ... Samples subjected to protease treatment were incubated on ice for 10 min with 100 μg/mL proteinase K (Sigma) before treatment with 1 mM PMSF for 10 min.

    Homogenization:

    Article Title: A short purification process for quantitative isolation of PrPSc from naturally occurring and experimental transmissible spongiform encephalopathies
    Article Snippet: For limiting dilution experiments, the polytron was replaced with the OmniGLH homogenizer (CAMLAB, UK) which worked equally well with disposable probes, a very useful factor to be considered while attempting multiple homogenizations with large number of samples. .. On the day of use, 100 μl of homogenate was diluted with an equal volume of the homogenization buffer and incubated with proteinase K (Sigma-Aldrich) for 1 hour at 37°C with mild rocking. .. Concentrations of proteinase K used varied with the species and are provided separately with each figure.

    Evaporation:

    Article Title: Identification of the active components in Bone Marrow Soup: a mitigator against irradiation-injury to salivary glands
    Article Snippet: Initially, four methods to deactivate BM Soup were tested. .. The micro-centrifuge tube cap was wrapped with parafilm to avoid evaporation; 2) Heated at 95 °C for 60 min; 3) Digested by Trypsin (1:20 w/w, T1426, Sigma-Aldrich, ST. Louis, USA) overnight at 37 °C; and 4) Digested by 0.1 μg/μl proteinase K (P2308 Sigma-Aldrich, ST. Louis, USA) overnight at 37 °C. .. Precipitates were removed by micro-centrifugation by 13,500 rpm for 15 min. 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, 120V, 60 min) and Coomassie blue staining were conducted to test the efficiency of deactivation.

    Acid Assay:

    Article Title: Identification of the active components in Bone Marrow Soup: a mitigator against irradiation-injury to salivary glands
    Article Snippet: The micro-centrifuge tube cap was wrapped with parafilm to avoid evaporation; 2) Heated at 95 °C for 60 min; 3) Digested by Trypsin (1:20 w/w, T1426, Sigma-Aldrich, ST. Louis, USA) overnight at 37 °C; and 4) Digested by 0.1 μg/μl proteinase K (P2308 Sigma-Aldrich, ST. Louis, USA) overnight at 37 °C. .. Precipitates were removed by micro-centrifugation by 13,500 rpm for 15 min. 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, 120V, 60 min) and Coomassie blue staining were conducted to test the efficiency of deactivation.

    Concentration Assay:

    Article Title: Rapid experimental SAD phasing and hot-spot identification with halogenated fragments
    Article Snippet: Prior to crystallization, the HIV-1 RT construct RT52A (20 mg ml−1 ; Bauman, Patel, Dharia et al. , 2013 ) was incubated with rilpivirine (TMC278/Edurant) at a 1:1.5 protein:drug molar ratio at room temperature (∼23°C) for 30 min. RT–rilpivirine crystals were produced in hanging drops at 4°C with a 1:1 ratio of protein solution and well solution consisting of 11%(v /v ) PEG 8000, 4%(v /v ) PEG 400, 50 mM imidazole pH 6.6, 10 mM spermine, 15 mM MgSO4 , 100 mM ammonium sulfate, 5 mM tris(2-carboxyethyl)phos­phine together with an experimentally optimized concentration of microseeds from previously generated and crushed RT–rilpivirine crystals (pre-seeding). .. Proteinase K was purchased from Sigma–Aldrich (St Louis, Missouri, USA) and crystallized as described previously (Beck et al. , 2010 ).

    Article Title: The PPE Domain of PPE17 Is Responsible for Its Surface Localization and Can Be Used to Express Heterologous Proteins on the Mycobacterial Surface
    Article Snippet: One aliquot was treated with proteinase K (Sigma-Aldrich) 100 µg ml−1 , whereas the other was left untreated and incubated for 30 min at 4°C. .. One aliquot was treated with proteinase K (Sigma-Aldrich) 100 µg ml−1 , whereas the other was left untreated and incubated for 30 min at 4°C.

    Article Title: The PPE Domain of PPE17 Is Responsible for Its Surface Localization and Can Be Used to Express Heterologous Proteins on the Mycobacterial Surface
    Article Snippet: Each sample was divided in two identical aliquots. .. One aliquot was treated with proteinase K (Sigma-Aldrich) up to a concentration of100 µg ml−1 , whereas the other was left untreated and incubated for 30 min at 4°C. .. The reaction was stopped by the addition of 1X complete EDTA-free protease inhibitor (Roche).

    Article Title: Molecular Analysis of Cases of Italian Sheep Scrapie and Comparison with Cases of Bovine Spongiform Encephalopathy (BSE) and Experimental BSE in Sheep
    Article Snippet: The homogenate was incubated for 20 min at room temperature and then centrifuged at 22,000 × g for 20 min (TLA 100.3 rotor; Beckman). .. Supernatant (1 ml) was added with 50 μl of a stock solution of proteinase K (Sigma) to give a final concentration of 50 μg/ml and then incubated for 1 h at 37°C with gentle shaking. .. Protease treatment was stopped with phenylmethylsulfonyl fluoride (PMSF) (Sigma) (30 μl of a stock solution, to give a final concentration of 3 mM).

    Fractionation:

    Article Title: The PPE Domain of PPE17 Is Responsible for Its Surface Localization and Can Be Used to Express Heterologous Proteins on the Mycobacterial Surface
    Article Snippet: One aliquot was treated with proteinase K (Sigma-Aldrich) up to a concentration of100 µg ml−1 , whereas the other was left untreated and incubated for 30 min at 4°C. .. The reaction was stopped by the addition of 1X complete EDTA-free protease inhibitor (Roche).

    Staining:

    Article Title: Dendritic Cell-derived Extracellular Vesicles mediate Mesenchymal Stem/Stromal Cell recruitment
    Article Snippet: Where indicated, EV and 100 K supernatant samples were incubated with proteinase K (10 μg/mL, 15 min, 37 °C, Sigma-Aldrich), for soluble proteins digestion, before or after EV lysis. .. Protein was quantified and the same amount (4 μg) resolved in gelatin-containing zymography gels as previously described , followed by gel incubation overnight with MMP substrate buffer (CaCl2 10 mM in Tris 50 mM buffer, pH7.5).

    T-Test:

    Article Title: Non-consensus heptamer sequences destabilize the RAG post-cleavage complex, making ends available to alternative DNA repair pathways
    Article Snippet: Reactions were then aliquoted into microfuge tubes and incubated at different temperatures, as indicated, or treated with stop buffer [10 mM Tris (pH 8.0), 10 mM EDTA, 0.2% SDS, 0.35 mg/ml proteinase K (Sigma Aldrich)] for 30 min and then ran out on 4–20% acrylamide TBE gels (Invitrogen). .. To generate the substrate used for the thermal stability experiments pECFP-289-NtCFP constructs with appropriate RSS mutations were digested with HpaI and NdeI (New England Biolabs) and a 1.6 Kb fragment was gel purified and then used for PCR amplification using NtCFP_upPCR 5′-CGCGCCGAGGTGAAGTTCGAGG-3′ and NtCFP_downPCR 5′-TGCCCCAGGATGTTGCCGTCCTCC-3′ to generate a 477 bp PCR product.

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    • granzyme b substrate acetyl  (Millipore)
    90
    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
    Granzyme B Substrate Acetyl, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 7 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

    Production of perforin, granzyme B and granulysin in CD8 + T cells as well as CD4 + T cells. ( A ) Enhanced production of perforin and granzyme B from CD8 + T cells cultured with LipoK stimulated M. leprae infected DCs. Intracellular staining of perforin and granzyme B was performed as follows: Cells were first stained with PE conjugated anti-CD4 or APC conjugated anti-CD8 mAb. Then, the cells were fixed in 2% formaldehyde, permeabilized in 0.1% saponin, and stained with FITC conjugated anti-perforin mAb or anti-granzyme B mAb or isotype control IgG2a. Figure shows the dot plot of the gated CD8 + T cells. The right hand quadrant shows CD8 high T cells (activated CD8 + T cells) and the number indicates the percentage of perforin or granzyme B positive T cells among gated CD8 high T cells. *To determine whether direct interaction between CD4 + and CD8 + T cells for perforin and granzyme B production from CD8 + T cells, is needed, CD4 + T cells were cultured in inserts in a 24-well plate, and were not allowed to interact directly with CD8 + T cells. As a control experiment, exogenous IL-2 (in the left hand dot plot) at a concentration of 50 U/ml was added to CD8 + T cells. ( B ) Enhanced expression of perforin and granzyme B from CD4 + T cells. The right hand quadrant shows CD4 high T cells, and the number indicates percentage of CD4 high T cells producing perforin and granzyme B. ( C ) Enhanced expression of granulysin from CD8 + and CD4 + T cells, co-cultured with LipoK and M. leprae stimulated DCs. The protocol was followed as per the staining of perforin, except that the surface stain used was FITC conjugated-CD4 and APC conjugated anti-CD8 mAb, and subsequently PE conjugated granulysin was used. Figure shows the dot plot of the gated CD8 + and CD4 + T cells. The right hand quadrant shows CD8 high or CD4 high T cells (activated T cells) and the number indicates the percentage of granulysin positive T cells among gated CD8 high and CD4 high T cells. Representative data of three separate experiments with different donors is shown.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: A Lipopeptide Facilitate Induction of Mycobacterium leprae Killing in Host Cells

    doi: 10.1371/journal.pntd.0001401

    Figure Lengend Snippet: Production of perforin, granzyme B and granulysin in CD8 + T cells as well as CD4 + T cells. ( A ) Enhanced production of perforin and granzyme B from CD8 + T cells cultured with LipoK stimulated M. leprae infected DCs. Intracellular staining of perforin and granzyme B was performed as follows: Cells were first stained with PE conjugated anti-CD4 or APC conjugated anti-CD8 mAb. Then, the cells were fixed in 2% formaldehyde, permeabilized in 0.1% saponin, and stained with FITC conjugated anti-perforin mAb or anti-granzyme B mAb or isotype control IgG2a. Figure shows the dot plot of the gated CD8 + T cells. The right hand quadrant shows CD8 high T cells (activated CD8 + T cells) and the number indicates the percentage of perforin or granzyme B positive T cells among gated CD8 high T cells. *To determine whether direct interaction between CD4 + and CD8 + T cells for perforin and granzyme B production from CD8 + T cells, is needed, CD4 + T cells were cultured in inserts in a 24-well plate, and were not allowed to interact directly with CD8 + T cells. As a control experiment, exogenous IL-2 (in the left hand dot plot) at a concentration of 50 U/ml was added to CD8 + T cells. ( B ) Enhanced expression of perforin and granzyme B from CD4 + T cells. The right hand quadrant shows CD4 high T cells, and the number indicates percentage of CD4 high T cells producing perforin and granzyme B. ( C ) Enhanced expression of granulysin from CD8 + and CD4 + T cells, co-cultured with LipoK and M. leprae stimulated DCs. The protocol was followed as per the staining of perforin, except that the surface stain used was FITC conjugated-CD4 and APC conjugated anti-CD8 mAb, and subsequently PE conjugated granulysin was used. Figure shows the dot plot of the gated CD8 + and CD4 + T cells. The right hand quadrant shows CD8 high or CD4 high T cells (activated T cells) and the number indicates the percentage of granulysin positive T cells among gated CD8 high and CD4 high T cells. Representative data of three separate experiments with different donors is shown.

    Article Snippet: To further determine whether the cytolytic granules namely, granulysin and granzyme B could directly kill M. leprae , the bacilli was incubated with human granulysin or granzyme B for a period of 3 days at 33°C.

    Techniques: Cell Culture, Infection, Staining, Concentration Assay, Expressing

    Reduction in the viability of M. leprae in DCs after co-culture with T cells and LipoK stimulation. ( A ) DCs were infected with M. leprae and stimulated with LipoK, 2 days later, cells were collected and the viability of M. leprae in DCs was measured by the radiorespirometric assay (metabolic CO 2 release) as described in Materials and Methods . In brief, 14 C labeled palmitic acid was added to the lysates of DCs and cultured at 33°C. After 7 days of culture, the amount of 14 CO 2 evolved was measured using a Packard 1500 TRI-CARB liquid scintillation analyzer. ( B ) DCs were infected with M. leprae as in A , and co-cultured with T cells. Six days after the co-culture, DCs were lysed, and the viability of M. leprae was determined by the radiorespirometric assay. ( C ) M. leprae at a concentration of 1×10 7 /well/200 µl in Middlebrook 7H9 media was incubated with granulysin or granzyme B for a period of 3 days at 33°C, and the viability determined as described in A . Unpaired Student's t test was used to find the statistical significance of the two sets of data. Representative data of three separate experiments is shown.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: A Lipopeptide Facilitate Induction of Mycobacterium leprae Killing in Host Cells

    doi: 10.1371/journal.pntd.0001401

    Figure Lengend Snippet: Reduction in the viability of M. leprae in DCs after co-culture with T cells and LipoK stimulation. ( A ) DCs were infected with M. leprae and stimulated with LipoK, 2 days later, cells were collected and the viability of M. leprae in DCs was measured by the radiorespirometric assay (metabolic CO 2 release) as described in Materials and Methods . In brief, 14 C labeled palmitic acid was added to the lysates of DCs and cultured at 33°C. After 7 days of culture, the amount of 14 CO 2 evolved was measured using a Packard 1500 TRI-CARB liquid scintillation analyzer. ( B ) DCs were infected with M. leprae as in A , and co-cultured with T cells. Six days after the co-culture, DCs were lysed, and the viability of M. leprae was determined by the radiorespirometric assay. ( C ) M. leprae at a concentration of 1×10 7 /well/200 µl in Middlebrook 7H9 media was incubated with granulysin or granzyme B for a period of 3 days at 33°C, and the viability determined as described in A . Unpaired Student's t test was used to find the statistical significance of the two sets of data. Representative data of three separate experiments is shown.

    Article Snippet: To further determine whether the cytolytic granules namely, granulysin and granzyme B could directly kill M. leprae , the bacilli was incubated with human granulysin or granzyme B for a period of 3 days at 33°C.

    Techniques: Co-Culture Assay, Infection, Labeling, Cell Culture, Concentration Assay, Incubation