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  • 95
    ATCC acanthamoeba
    Protease zymogram for the <t>Acanthamoeba</t> T5 isolate. Lanes: ( a ) Acanthamoeba conditioned medium (ACM), ( b ) ACM incubated with phenylmethylsulfonyl fluoride (PMSF) (inhibitor of serine proteases), ( c ) ACM incubated with 2-iodoacetamide (inhibitor of cysteine proteases), ( d ) crude extract of trophozoites, ( e ) crude extract of trophozoites incubated with PMSF and ( f ) crude extract of trophozoites incubated with 2-iodoacetamide.
    Acanthamoeba, supplied by ATCC, used in various techniques. Bioz Stars score: 95/100, based on 119 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore 5a2
    Protease zymogram for the <t>Acanthamoeba</t> T5 isolate. Lanes: ( a ) Acanthamoeba conditioned medium (ACM), ( b ) ACM incubated with phenylmethylsulfonyl fluoride (PMSF) (inhibitor of serine proteases), ( c ) ACM incubated with 2-iodoacetamide (inhibitor of cysteine proteases), ( d ) crude extract of trophozoites, ( e ) crude extract of trophozoites incubated with PMSF and ( f ) crude extract of trophozoites incubated with 2-iodoacetamide.
    5a2, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Pharmingen jes10 5a2
    Protease zymogram for the <t>Acanthamoeba</t> T5 isolate. Lanes: ( a ) Acanthamoeba conditioned medium (ACM), ( b ) ACM incubated with phenylmethylsulfonyl fluoride (PMSF) (inhibitor of serine proteases), ( c ) ACM incubated with 2-iodoacetamide (inhibitor of cysteine proteases), ( d ) crude extract of trophozoites, ( e ) crude extract of trophozoites incubated with PMSF and ( f ) crude extract of trophozoites incubated with 2-iodoacetamide.
    Jes10 5a2, supplied by Pharmingen, used in various techniques. Bioz Stars score: 85/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson jes10 5a2
    Protease zymogram for the <t>Acanthamoeba</t> T5 isolate. Lanes: ( a ) Acanthamoeba conditioned medium (ACM), ( b ) ACM incubated with phenylmethylsulfonyl fluoride (PMSF) (inhibitor of serine proteases), ( c ) ACM incubated with 2-iodoacetamide (inhibitor of cysteine proteases), ( d ) crude extract of trophozoites, ( e ) crude extract of trophozoites incubated with PMSF and ( f ) crude extract of trophozoites incubated with 2-iodoacetamide.
    Jes10 5a2, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 85/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Merck KGaA 5a2
    Protease zymogram for the <t>Acanthamoeba</t> T5 isolate. Lanes: ( a ) Acanthamoeba conditioned medium (ACM), ( b ) ACM incubated with phenylmethylsulfonyl fluoride (PMSF) (inhibitor of serine proteases), ( c ) ACM incubated with 2-iodoacetamide (inhibitor of cysteine proteases), ( d ) crude extract of trophozoites, ( e ) crude extract of trophozoites incubated with PMSF and ( f ) crude extract of trophozoites incubated with 2-iodoacetamide.
    5a2, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 93/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher 5a2
    Frequencies of splenic <t>IL-13</t> <t>mRNA</t> + cells following stimulation with PMA/ionomycin or H. meleagridis . A , B Representative pseudocolor plots show IL-13 mRNA versus CD4 expression in pre-gated (not depicted) total live splenocytes isolated from birds 2 weeks pi and 5 weeks pi. Approximately 320 000 lymphocytes are shown in each plot and numbers indicate frequencies of IL-13 mRNA + cells within total live splenocytes. Graphs on the right display frequencies of IL-13 mRNA + live lymphocytes from all birds. Each symbol represents one bird, black and red colored symbols represent birds sacrificed 2 weeks pi and 5 weeks pi, respectively. A Scatter plots show percent of IL-13 mRNA + cells within live lymphocytes after PMA/ionomycin stimulation compared to medium in control and infected birds (left panel). Right panel: comparison of IL-13 mRNA + lymphocyte frequencies after stimulation with PMA/ionomycin between infected and control birds. B Scatter plots as in A but after H. meleagridis / E. coli stimulation and E. coli -only stimulation. Right panel shows in addition percent of IL-13 mRNA + cells after E. coli correction for infected and control birds. Asterisks indicate p -value: * p ≤ 0.05.
    5a2, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 326 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Trellis Bioscience 5a2
    Frequencies of splenic <t>IL-13</t> <t>mRNA</t> + cells following stimulation with PMA/ionomycin or H. meleagridis . A , B Representative pseudocolor plots show IL-13 mRNA versus CD4 expression in pre-gated (not depicted) total live splenocytes isolated from birds 2 weeks pi and 5 weeks pi. Approximately 320 000 lymphocytes are shown in each plot and numbers indicate frequencies of IL-13 mRNA + cells within total live splenocytes. Graphs on the right display frequencies of IL-13 mRNA + live lymphocytes from all birds. Each symbol represents one bird, black and red colored symbols represent birds sacrificed 2 weeks pi and 5 weeks pi, respectively. A Scatter plots show percent of IL-13 mRNA + cells within live lymphocytes after PMA/ionomycin stimulation compared to medium in control and infected birds (left panel). Right panel: comparison of IL-13 mRNA + lymphocyte frequencies after stimulation with PMA/ionomycin between infected and control birds. B Scatter plots as in A but after H. meleagridis / E. coli stimulation and E. coli -only stimulation. Right panel shows in addition percent of IL-13 mRNA + cells after E. coli correction for infected and control birds. Asterisks indicate p -value: * p ≤ 0.05.
    5a2, supplied by Trellis Bioscience, used in various techniques. Bioz Stars score: 88/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam anti eif 5a2
    Generation of a conditional <t>eIF-5A2</t> knock-out mouse strain using an ES cell clone harboring a targeted mutation of the eIF-5A2 gene. A, schematic representation of the knock-out strategy for achieving a conditional knock-out of the eIF-5A2 gene. The two
    Anti Eif 5a2, supplied by Abcam, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson anti il 13 pycoerythrin jes10 5a2
    Generation of a conditional <t>eIF-5A2</t> knock-out mouse strain using an ES cell clone harboring a targeted mutation of the eIF-5A2 gene. A, schematic representation of the knock-out strategy for achieving a conditional knock-out of the eIF-5A2 gene. The two
    Anti Il 13 Pycoerythrin Jes10 5a2, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 88/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti pd l1
    Generation of a conditional <t>eIF-5A2</t> knock-out mouse strain using an ES cell clone harboring a targeted mutation of the eIF-5A2 gene. A, schematic representation of the knock-out strategy for achieving a conditional knock-out of the eIF-5A2 gene. The two
    Anti Pd L1, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 38 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam eif 5a2 mouse monoclonal abcam ab57421
    Generation of a conditional <t>eIF-5A2</t> knock-out mouse strain using an ES cell clone harboring a targeted mutation of the eIF-5A2 gene. A, schematic representation of the knock-out strategy for achieving a conditional knock-out of the eIF-5A2 gene. The two
    Eif 5a2 Mouse Monoclonal Abcam Ab57421, supplied by Abcam, used in various techniques. Bioz Stars score: 85/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher eif 5a2
    Skeletal aging phenotypes in <t>eIF-5A2</t> transgenic mice . (A) X-ray radiograph was used to examine skeletal changes in 24-week-old eIF-5A2 transgenic mouse (upper) and their wild-type sibling (lower). Kyphosis was observed in eIF5A2 mouse (indicated by an arrow). ( B ) Representative radiograph of the density of femur bone in an eIF-5A2 transgenic mouse (24-week-old) and a wild-type mouse. ( C, D ) Alcian blue and Alizarin red staining of skulls from a 2-week-old transgenic mouse (upper) and its wild-type sibling (lower). A wider cranial sutures ( C ) and fontanelle ( D ) were observed in transgenic pup (indicated by arrows). ( E ) Representative skeletal staining of hind limb in a 2-week-old transgenic mouse (upper) and its wild-type sibling (lower). A clear ossification of patella was observed in the knee joint of wild-type mouse but not in the transgenic mouse (indicated by arrows).
    Eif 5a2, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher containing neutralizing antibody against il 13
    Skeletal aging phenotypes in <t>eIF-5A2</t> transgenic mice . (A) X-ray radiograph was used to examine skeletal changes in 24-week-old eIF-5A2 transgenic mouse (upper) and their wild-type sibling (lower). Kyphosis was observed in eIF5A2 mouse (indicated by an arrow). ( B ) Representative radiograph of the density of femur bone in an eIF-5A2 transgenic mouse (24-week-old) and a wild-type mouse. ( C, D ) Alcian blue and Alizarin red staining of skulls from a 2-week-old transgenic mouse (upper) and its wild-type sibling (lower). A wider cranial sutures ( C ) and fontanelle ( D ) were observed in transgenic pup (indicated by arrows). ( E ) Representative skeletal staining of hind limb in a 2-week-old transgenic mouse (upper) and its wild-type sibling (lower). A clear ossification of patella was observed in the knee joint of wild-type mouse but not in the transgenic mouse (indicated by arrows).
    Containing Neutralizing Antibody Against Il 13, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Pharmingen il 13 jes10 5a2
    Skeletal aging phenotypes in <t>eIF-5A2</t> transgenic mice . (A) X-ray radiograph was used to examine skeletal changes in 24-week-old eIF-5A2 transgenic mouse (upper) and their wild-type sibling (lower). Kyphosis was observed in eIF5A2 mouse (indicated by an arrow). ( B ) Representative radiograph of the density of femur bone in an eIF-5A2 transgenic mouse (24-week-old) and a wild-type mouse. ( C, D ) Alcian blue and Alizarin red staining of skulls from a 2-week-old transgenic mouse (upper) and its wild-type sibling (lower). A wider cranial sutures ( C ) and fontanelle ( D ) were observed in transgenic pup (indicated by arrows). ( E ) Representative skeletal staining of hind limb in a 2-week-old transgenic mouse (upper) and its wild-type sibling (lower). A clear ossification of patella was observed in the knee joint of wild-type mouse but not in the transgenic mouse (indicated by arrows).
    Il 13 Jes10 5a2, supplied by Pharmingen, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson pe anti il 13 jes10 5a2 mabs
    Skeletal aging phenotypes in <t>eIF-5A2</t> transgenic mice . (A) X-ray radiograph was used to examine skeletal changes in 24-week-old eIF-5A2 transgenic mouse (upper) and their wild-type sibling (lower). Kyphosis was observed in eIF5A2 mouse (indicated by an arrow). ( B ) Representative radiograph of the density of femur bone in an eIF-5A2 transgenic mouse (24-week-old) and a wild-type mouse. ( C, D ) Alcian blue and Alizarin red staining of skulls from a 2-week-old transgenic mouse (upper) and its wild-type sibling (lower). A wider cranial sutures ( C ) and fontanelle ( D ) were observed in transgenic pup (indicated by arrows). ( E ) Representative skeletal staining of hind limb in a 2-week-old transgenic mouse (upper) and its wild-type sibling (lower). A clear ossification of patella was observed in the knee joint of wild-type mouse but not in the transgenic mouse (indicated by arrows).
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    Image Search Results


    Protease zymogram for the Acanthamoeba T5 isolate. Lanes: ( a ) Acanthamoeba conditioned medium (ACM), ( b ) ACM incubated with phenylmethylsulfonyl fluoride (PMSF) (inhibitor of serine proteases), ( c ) ACM incubated with 2-iodoacetamide (inhibitor of cysteine proteases), ( d ) crude extract of trophozoites, ( e ) crude extract of trophozoites incubated with PMSF and ( f ) crude extract of trophozoites incubated with 2-iodoacetamide.

    Journal: Pathogens

    Article Title: Isolation of Acanthamoeba T5 from Water: Characterization of Its Pathogenic Potential, Including the Production of Extracellular Vesicles

    doi: 10.3390/pathogens9020144

    Figure Lengend Snippet: Protease zymogram for the Acanthamoeba T5 isolate. Lanes: ( a ) Acanthamoeba conditioned medium (ACM), ( b ) ACM incubated with phenylmethylsulfonyl fluoride (PMSF) (inhibitor of serine proteases), ( c ) ACM incubated with 2-iodoacetamide (inhibitor of cysteine proteases), ( d ) crude extract of trophozoites, ( e ) crude extract of trophozoites incubated with PMSF and ( f ) crude extract of trophozoites incubated with 2-iodoacetamide.

    Article Snippet: Evaluation of the in Vitro Effect of Acanthamoeba in Cell Cultures Cell culture: Madin–Darby canine kidney (MDCK) epithelial cells (NBL2 ATCC CCL-34TM) and Vero cells (ATCC CCL-81) were grown in 75 cm2 cell culture flasks (Corning, Corning Incorporated, NY, USA) with RPMI 1640 medium (Gibco GranIsland, NY, USA) supplemented with penicillin (100 U/mL), streptomycin (100 pg/mL) and 10% fetal calf serum (Gibco, Gran Island, NY, USA).

    Techniques: Incubation

    MDCK cell monolayers stained with the fluorescent stain Hoescht 33342 and incubated with Acanthamoeba. Images of the monolayer at 0 and 24 h postincubation with Acanthamoeba CLC-16 (genotype T3) (positive control of cytopathic effect) and Acanthamoeba genotype T5 are shown. Arrows indicate possible pyknotic cells.

    Journal: Pathogens

    Article Title: Isolation of Acanthamoeba T5 from Water: Characterization of Its Pathogenic Potential, Including the Production of Extracellular Vesicles

    doi: 10.3390/pathogens9020144

    Figure Lengend Snippet: MDCK cell monolayers stained with the fluorescent stain Hoescht 33342 and incubated with Acanthamoeba. Images of the monolayer at 0 and 24 h postincubation with Acanthamoeba CLC-16 (genotype T3) (positive control of cytopathic effect) and Acanthamoeba genotype T5 are shown. Arrows indicate possible pyknotic cells.

    Article Snippet: Evaluation of the in Vitro Effect of Acanthamoeba in Cell Cultures Cell culture: Madin–Darby canine kidney (MDCK) epithelial cells (NBL2 ATCC CCL-34TM) and Vero cells (ATCC CCL-81) were grown in 75 cm2 cell culture flasks (Corning, Corning Incorporated, NY, USA) with RPMI 1640 medium (Gibco GranIsland, NY, USA) supplemented with penicillin (100 U/mL), streptomycin (100 pg/mL) and 10% fetal calf serum (Gibco, Gran Island, NY, USA).

    Techniques: Staining, Incubation, Positive Control

    Atomic force microscopy Z-height images and nanomechanical maps showing stiffness, Young modulus and adhesion profile of the EVs of Acanthamoeba T5 incubated at 28 °C ( A ) and 37 °C ( B ) in PYG medium.

    Journal: Pathogens

    Article Title: Isolation of Acanthamoeba T5 from Water: Characterization of Its Pathogenic Potential, Including the Production of Extracellular Vesicles

    doi: 10.3390/pathogens9020144

    Figure Lengend Snippet: Atomic force microscopy Z-height images and nanomechanical maps showing stiffness, Young modulus and adhesion profile of the EVs of Acanthamoeba T5 incubated at 28 °C ( A ) and 37 °C ( B ) in PYG medium.

    Article Snippet: Evaluation of the in Vitro Effect of Acanthamoeba in Cell Cultures Cell culture: Madin–Darby canine kidney (MDCK) epithelial cells (NBL2 ATCC CCL-34TM) and Vero cells (ATCC CCL-81) were grown in 75 cm2 cell culture flasks (Corning, Corning Incorporated, NY, USA) with RPMI 1640 medium (Gibco GranIsland, NY, USA) supplemented with penicillin (100 U/mL), streptomycin (100 pg/mL) and 10% fetal calf serum (Gibco, Gran Island, NY, USA).

    Techniques: Microscopy, Incubation

    Protease zymogram of the extracellular vesicles (EVs) of the Acanthamoeba T5 isolate. For each lane, 8 µL of the sample at a concentration 1.1 µg/µL was loaded onto the gel. Lanes: 1) EVs of amoebae incubated at 28 °C, 2) EVs of amoebae incubated at 37 °C, 3) ACM of amoebae incubated at 28 °C and 4) ACM of amoebae incubated at 37 °C.

    Journal: Pathogens

    Article Title: Isolation of Acanthamoeba T5 from Water: Characterization of Its Pathogenic Potential, Including the Production of Extracellular Vesicles

    doi: 10.3390/pathogens9020144

    Figure Lengend Snippet: Protease zymogram of the extracellular vesicles (EVs) of the Acanthamoeba T5 isolate. For each lane, 8 µL of the sample at a concentration 1.1 µg/µL was loaded onto the gel. Lanes: 1) EVs of amoebae incubated at 28 °C, 2) EVs of amoebae incubated at 37 °C, 3) ACM of amoebae incubated at 28 °C and 4) ACM of amoebae incubated at 37 °C.

    Article Snippet: Evaluation of the in Vitro Effect of Acanthamoeba in Cell Cultures Cell culture: Madin–Darby canine kidney (MDCK) epithelial cells (NBL2 ATCC CCL-34TM) and Vero cells (ATCC CCL-81) were grown in 75 cm2 cell culture flasks (Corning, Corning Incorporated, NY, USA) with RPMI 1640 medium (Gibco GranIsland, NY, USA) supplemented with penicillin (100 U/mL), streptomycin (100 pg/mL) and 10% fetal calf serum (Gibco, Gran Island, NY, USA).

    Techniques: Concentration Assay, Incubation

    Crystal violet stain that shows the cytopathic effect of Acanthamoeba T5 over Madin-Darby canine kidney (MDCK) and Vero cells. Amoebae were incubated with MDCK or Vero cells in 24-well plates for 24 h at 37 °C and their cytopathic effect was observed using the crystal violet stain. Lanes: ( A ) MDCK cell control, ( B ) MDCK cells incubated with Acanthamoeba CLC-16 (control strain of cytopathic effect), ( C ) MDCK cells incubated with Acanthamoeba T5; ( D ) Vero cell control, ( E ) Vero cells incubated with Acanthamoeba Neff (control strain of cytopathic effect) and ( F ) Vero cells incubated with Acanthamoeba T5. Images are representative of experiments performed in triplicate.

    Journal: Pathogens

    Article Title: Isolation of Acanthamoeba T5 from Water: Characterization of Its Pathogenic Potential, Including the Production of Extracellular Vesicles

    doi: 10.3390/pathogens9020144

    Figure Lengend Snippet: Crystal violet stain that shows the cytopathic effect of Acanthamoeba T5 over Madin-Darby canine kidney (MDCK) and Vero cells. Amoebae were incubated with MDCK or Vero cells in 24-well plates for 24 h at 37 °C and their cytopathic effect was observed using the crystal violet stain. Lanes: ( A ) MDCK cell control, ( B ) MDCK cells incubated with Acanthamoeba CLC-16 (control strain of cytopathic effect), ( C ) MDCK cells incubated with Acanthamoeba T5; ( D ) Vero cell control, ( E ) Vero cells incubated with Acanthamoeba Neff (control strain of cytopathic effect) and ( F ) Vero cells incubated with Acanthamoeba T5. Images are representative of experiments performed in triplicate.

    Article Snippet: Evaluation of the in Vitro Effect of Acanthamoeba in Cell Cultures Cell culture: Madin–Darby canine kidney (MDCK) epithelial cells (NBL2 ATCC CCL-34TM) and Vero cells (ATCC CCL-81) were grown in 75 cm2 cell culture flasks (Corning, Corning Incorporated, NY, USA) with RPMI 1640 medium (Gibco GranIsland, NY, USA) supplemented with penicillin (100 U/mL), streptomycin (100 pg/mL) and 10% fetal calf serum (Gibco, Gran Island, NY, USA).

    Techniques: Staining, Incubation

    Ultrastructure of “ Ca . Amoebophilus asiaticus” 5a2 within its Acanthamoeba host cell. (A) Transmission electron micrograph showing the distribution of “ Ca . Amoebophilus asiaticus” in its Acanthamoeba host cell. (B and C)

    Journal: Journal of Bacteriology

    Article Title: The Genome of the Amoeba Symbiont “Candidatus Amoebophilus asiaticus” Reveals Common Mechanisms for Host Cell Interaction among Amoeba-Associated Bacteria

    doi: 10.1128/JB.01379-09

    Figure Lengend Snippet: Ultrastructure of “ Ca . Amoebophilus asiaticus” 5a2 within its Acanthamoeba host cell. (A) Transmission electron micrograph showing the distribution of “ Ca . Amoebophilus asiaticus” in its Acanthamoeba host cell. (B and C)

    Article Snippet: Amoebophilus asiaticus” 5a2 (ATCC number PRA-228) was isolated from lake sediment in Austria ( ) and cultivated using peptone-yeast-glucose-medium (PYG) [containing 20 g/liter proteose peptone, 18 g/liter glucose, 2 g/liter yeast extract, 3.4 mM sodium citrate-dihydrate, 4 mM MgSO4 ·7H2 O, 2 mM Na2 HPO4 ·2H2 O, 1.7 mM KH2 PO4 , 0.05 mM Fe(NH4 )2 (SO4 )2 ·6H2 O] as described previously ( ).

    Techniques: Transmission Assay

    Transcription of IS elements during intracellular growth of A. asiaticus 5a2 in its Acanthamoeba host . Transcription of 13 selected A. asiaticus IS elements was analyzed with reverse transcriptase PCR. Whole RNA from the Acanthamoeba host harboring A. asiaticus was transcribed into cDNA and subsequently used for PCR. (A) Reverse transcriptase PCR reactions. Lanes 1: cDNA; lanes 2: positive control, genomic DNA purified from amoebae containing A. asiaticus ; lanes 3: negative control, no nucleic acids added. (B) PCR using 16S rRNA gene-specific primers was used to control for the absence of DNA in the RNA preparation. Lane 4: positive control, genomic DNA; lane 5: RNA; lane 6: negative control, no nucleic acids added. m: molecular size marker. Reverse transcriptase-PCR reactions were performed in three biological independent replicates.

    Journal: BMC Evolutionary Biology

    Article Title: A bacterial genome in transition - an exceptional enrichment of IS elements but lack of evidence for recent transposition in the symbiont Amoebophilus asiaticus

    doi: 10.1186/1471-2148-11-270

    Figure Lengend Snippet: Transcription of IS elements during intracellular growth of A. asiaticus 5a2 in its Acanthamoeba host . Transcription of 13 selected A. asiaticus IS elements was analyzed with reverse transcriptase PCR. Whole RNA from the Acanthamoeba host harboring A. asiaticus was transcribed into cDNA and subsequently used for PCR. (A) Reverse transcriptase PCR reactions. Lanes 1: cDNA; lanes 2: positive control, genomic DNA purified from amoebae containing A. asiaticus ; lanes 3: negative control, no nucleic acids added. (B) PCR using 16S rRNA gene-specific primers was used to control for the absence of DNA in the RNA preparation. Lane 4: positive control, genomic DNA; lane 5: RNA; lane 6: negative control, no nucleic acids added. m: molecular size marker. Reverse transcriptase-PCR reactions were performed in three biological independent replicates.

    Article Snippet: Cultivation and isolation of amoebae Amoebae harboring A. asiaticus 5a2 (ATCC no. PRA-228) and amoebae harboring A. asiaticus EIDS3 (ATCC no. PRA-221) were maintained as adherent culture in 25 cm2 tissue culture flasks containing 10 ml peptone-yeast-glucose medium (PYG: 20 g/l proteose peptone, 2 g/l yeast extract, 90 mM glucose, 4 mM MgSO4 *7H2 O, 3.4 mM C6 H5 Na3 O7 *2H2 O, 2.5 mM KH2 PO4 , 1.3 mM Na2 HPO4 *2H2 O, 51 μM Fe(NH4 )2 (SO4 )2 *6H2 O).

    Techniques: Polymerase Chain Reaction, Positive Control, Purification, Negative Control, Marker

    Contiguous transcription of A. asiaticus IS elements with their downstream genes . Transcription was analyzed with reverse transcriptase PCR. Whole RNA from the Acanthamoeba host and A. asiaticus 5a2 was transcribed into cDNA and subsequently used for PCR. Genomic organization of the tested loci and the expected sizes of the PCR products are shown in Figure 4. Locus_tags are indicated above the gel images; lanes 1: cDNA; lanes 2: positive control, genomic DNA; lanes 3: negative control, no nucleic acids added. Contiguous transcription was demonstrated for all tested loci except for the IS element Aasi_0897 (ISCaa16) and its downstream gene Aasi_1745. The genes Aasi_1200/1201 were used as negative control (as they are located on different DNA strands and have opposing orientation). The groEL/groES operon (Aasi_0308/0309) was used as a positive control. All experiments were performed in three biological independent replicates.

    Journal: BMC Evolutionary Biology

    Article Title: A bacterial genome in transition - an exceptional enrichment of IS elements but lack of evidence for recent transposition in the symbiont Amoebophilus asiaticus

    doi: 10.1186/1471-2148-11-270

    Figure Lengend Snippet: Contiguous transcription of A. asiaticus IS elements with their downstream genes . Transcription was analyzed with reverse transcriptase PCR. Whole RNA from the Acanthamoeba host and A. asiaticus 5a2 was transcribed into cDNA and subsequently used for PCR. Genomic organization of the tested loci and the expected sizes of the PCR products are shown in Figure 4. Locus_tags are indicated above the gel images; lanes 1: cDNA; lanes 2: positive control, genomic DNA; lanes 3: negative control, no nucleic acids added. Contiguous transcription was demonstrated for all tested loci except for the IS element Aasi_0897 (ISCaa16) and its downstream gene Aasi_1745. The genes Aasi_1200/1201 were used as negative control (as they are located on different DNA strands and have opposing orientation). The groEL/groES operon (Aasi_0308/0309) was used as a positive control. All experiments were performed in three biological independent replicates.

    Article Snippet: Cultivation and isolation of amoebae Amoebae harboring A. asiaticus 5a2 (ATCC no. PRA-228) and amoebae harboring A. asiaticus EIDS3 (ATCC no. PRA-221) were maintained as adherent culture in 25 cm2 tissue culture flasks containing 10 ml peptone-yeast-glucose medium (PYG: 20 g/l proteose peptone, 2 g/l yeast extract, 90 mM glucose, 4 mM MgSO4 *7H2 O, 3.4 mM C6 H5 Na3 O7 *2H2 O, 2.5 mM KH2 PO4 , 1.3 mM Na2 HPO4 *2H2 O, 51 μM Fe(NH4 )2 (SO4 )2 *6H2 O).

    Techniques: Polymerase Chain Reaction, Positive Control, Negative Control

    Analysis of transpositional activity of the most abundant IS elements of A. asiaticus 5a2 . Transposition of IS elements was analyzed with Southern hybridizations using IS element-specific probes and DNA purified from the same A. asiaticus 5a2 culture in November 2006 and July 2009, respectively. DNA was digested with Eco32I (except for ISCaa2, where HindIII was used). Each visible band corresponds to at least one IS element copy on the respective DNA fragment, as the restriction endonucleases do not cut within the IS elements. IS elements are indicated above each hybridization; lanes 1: DNA isolated November 2006; lanes 2: DNA isolated July 2009. The absence of changes in the banding patterns between both time points indicates that no (major) chromosomal rearrangements due to IS element transposition has occurred.

    Journal: BMC Evolutionary Biology

    Article Title: A bacterial genome in transition - an exceptional enrichment of IS elements but lack of evidence for recent transposition in the symbiont Amoebophilus asiaticus

    doi: 10.1186/1471-2148-11-270

    Figure Lengend Snippet: Analysis of transpositional activity of the most abundant IS elements of A. asiaticus 5a2 . Transposition of IS elements was analyzed with Southern hybridizations using IS element-specific probes and DNA purified from the same A. asiaticus 5a2 culture in November 2006 and July 2009, respectively. DNA was digested with Eco32I (except for ISCaa2, where HindIII was used). Each visible band corresponds to at least one IS element copy on the respective DNA fragment, as the restriction endonucleases do not cut within the IS elements. IS elements are indicated above each hybridization; lanes 1: DNA isolated November 2006; lanes 2: DNA isolated July 2009. The absence of changes in the banding patterns between both time points indicates that no (major) chromosomal rearrangements due to IS element transposition has occurred.

    Article Snippet: Cultivation and isolation of amoebae Amoebae harboring A. asiaticus 5a2 (ATCC no. PRA-228) and amoebae harboring A. asiaticus EIDS3 (ATCC no. PRA-221) were maintained as adherent culture in 25 cm2 tissue culture flasks containing 10 ml peptone-yeast-glucose medium (PYG: 20 g/l proteose peptone, 2 g/l yeast extract, 90 mM glucose, 4 mM MgSO4 *7H2 O, 3.4 mM C6 H5 Na3 O7 *2H2 O, 2.5 mM KH2 PO4 , 1.3 mM Na2 HPO4 *2H2 O, 51 μM Fe(NH4 )2 (SO4 )2 *6H2 O).

    Techniques: Activity Assay, Purification, Hybridization, Isolation

    Frequencies of splenic IL-13 mRNA + cells following stimulation with PMA/ionomycin or H. meleagridis . A , B Representative pseudocolor plots show IL-13 mRNA versus CD4 expression in pre-gated (not depicted) total live splenocytes isolated from birds 2 weeks pi and 5 weeks pi. Approximately 320 000 lymphocytes are shown in each plot and numbers indicate frequencies of IL-13 mRNA + cells within total live splenocytes. Graphs on the right display frequencies of IL-13 mRNA + live lymphocytes from all birds. Each symbol represents one bird, black and red colored symbols represent birds sacrificed 2 weeks pi and 5 weeks pi, respectively. A Scatter plots show percent of IL-13 mRNA + cells within live lymphocytes after PMA/ionomycin stimulation compared to medium in control and infected birds (left panel). Right panel: comparison of IL-13 mRNA + lymphocyte frequencies after stimulation with PMA/ionomycin between infected and control birds. B Scatter plots as in A but after H. meleagridis / E. coli stimulation and E. coli -only stimulation. Right panel shows in addition percent of IL-13 mRNA + cells after E. coli correction for infected and control birds. Asterisks indicate p -value: * p ≤ 0.05.

    Journal: Veterinary Research

    Article Title: Cytokine production and phenotype of Histomonas meleagridis-specific T cells in the chicken

    doi: 10.1186/s13567-019-0726-z

    Figure Lengend Snippet: Frequencies of splenic IL-13 mRNA + cells following stimulation with PMA/ionomycin or H. meleagridis . A , B Representative pseudocolor plots show IL-13 mRNA versus CD4 expression in pre-gated (not depicted) total live splenocytes isolated from birds 2 weeks pi and 5 weeks pi. Approximately 320 000 lymphocytes are shown in each plot and numbers indicate frequencies of IL-13 mRNA + cells within total live splenocytes. Graphs on the right display frequencies of IL-13 mRNA + live lymphocytes from all birds. Each symbol represents one bird, black and red colored symbols represent birds sacrificed 2 weeks pi and 5 weeks pi, respectively. A Scatter plots show percent of IL-13 mRNA + cells within live lymphocytes after PMA/ionomycin stimulation compared to medium in control and infected birds (left panel). Right panel: comparison of IL-13 mRNA + lymphocyte frequencies after stimulation with PMA/ionomycin between infected and control birds. B Scatter plots as in A but after H. meleagridis / E. coli stimulation and E. coli -only stimulation. Right panel shows in addition percent of IL-13 mRNA + cells after E. coli correction for infected and control birds. Asterisks indicate p -value: * p ≤ 0.05.

    Article Snippet: In our study, detection of IFN-γ by ICS was tested alongside to detection of IL-13 mRNA by PrimeFlow™ RNA Assay (Thermo Fisher Scientific).

    Techniques: Expressing, Isolation, Infection

    Generation of a conditional eIF-5A2 knock-out mouse strain using an ES cell clone harboring a targeted mutation of the eIF-5A2 gene. A, schematic representation of the knock-out strategy for achieving a conditional knock-out of the eIF-5A2 gene. The two

    Journal: The Journal of Biological Chemistry

    Article Title: Biological Relevance and Therapeutic Potential of the Hypusine Modification System *

    doi: 10.1074/jbc.M115.664490

    Figure Lengend Snippet: Generation of a conditional eIF-5A2 knock-out mouse strain using an ES cell clone harboring a targeted mutation of the eIF-5A2 gene. A, schematic representation of the knock-out strategy for achieving a conditional knock-out of the eIF-5A2 gene. The two

    Article Snippet: One- and two-dimensional Western blot analyses were carried out as described before ( , ) using anti-eIF-5A1 (Novus Biologicals, Littleton, CO) and anti-eIF-5A2 (Abcam, Cambridge, UK).

    Techniques: Knock-Out, Mutagenesis

    eIF-5A2 knock-out mice are viable and fertile. A, offspring analysis of the indicated breedings. B, Kaplan-Meier plot showing overall survival of mice at the indicated time points after birth. C, mRNA levels of eIF-5A2 in different tissues of wild type

    Journal: The Journal of Biological Chemistry

    Article Title: Biological Relevance and Therapeutic Potential of the Hypusine Modification System *

    doi: 10.1074/jbc.M115.664490

    Figure Lengend Snippet: eIF-5A2 knock-out mice are viable and fertile. A, offspring analysis of the indicated breedings. B, Kaplan-Meier plot showing overall survival of mice at the indicated time points after birth. C, mRNA levels of eIF-5A2 in different tissues of wild type

    Article Snippet: One- and two-dimensional Western blot analyses were carried out as described before ( , ) using anti-eIF-5A1 (Novus Biologicals, Littleton, CO) and anti-eIF-5A2 (Abcam, Cambridge, UK).

    Techniques: Knock-Out, Mouse Assay

    Skeletal aging phenotypes in eIF-5A2 transgenic mice . (A) X-ray radiograph was used to examine skeletal changes in 24-week-old eIF-5A2 transgenic mouse (upper) and their wild-type sibling (lower). Kyphosis was observed in eIF5A2 mouse (indicated by an arrow). ( B ) Representative radiograph of the density of femur bone in an eIF-5A2 transgenic mouse (24-week-old) and a wild-type mouse. ( C, D ) Alcian blue and Alizarin red staining of skulls from a 2-week-old transgenic mouse (upper) and its wild-type sibling (lower). A wider cranial sutures ( C ) and fontanelle ( D ) were observed in transgenic pup (indicated by arrows). ( E ) Representative skeletal staining of hind limb in a 2-week-old transgenic mouse (upper) and its wild-type sibling (lower). A clear ossification of patella was observed in the knee joint of wild-type mouse but not in the transgenic mouse (indicated by arrows).

    Journal: BMC Cancer

    Article Title: Overexpression of eIF-5A2 in mice causes accelerated organismal aging by increasing chromosome instability

    doi: 10.1186/1471-2407-11-199

    Figure Lengend Snippet: Skeletal aging phenotypes in eIF-5A2 transgenic mice . (A) X-ray radiograph was used to examine skeletal changes in 24-week-old eIF-5A2 transgenic mouse (upper) and their wild-type sibling (lower). Kyphosis was observed in eIF5A2 mouse (indicated by an arrow). ( B ) Representative radiograph of the density of femur bone in an eIF-5A2 transgenic mouse (24-week-old) and a wild-type mouse. ( C, D ) Alcian blue and Alizarin red staining of skulls from a 2-week-old transgenic mouse (upper) and its wild-type sibling (lower). A wider cranial sutures ( C ) and fontanelle ( D ) were observed in transgenic pup (indicated by arrows). ( E ) Representative skeletal staining of hind limb in a 2-week-old transgenic mouse (upper) and its wild-type sibling (lower). A clear ossification of patella was observed in the knee joint of wild-type mouse but not in the transgenic mouse (indicated by arrows).

    Article Snippet: About 10 μg of lysate was separated by SDS-polyacrylamide gel electrophoresis, transferred to a PVDF Hybond-P membrane (Amersham Pharmacia Biotechnology, Piscataway, NJ), and detected by antibodies for eIF-5A2 (a mouse monoclonal antibody raised against the 54 residues of eIF-5A2), p53 (Zymed, San Francisco, CA), p21 (Upstate, Temecula, CA), p19 (Upstate, Lake Placid, NY), CDK4 (Cell Signaling Technology, Beverley, MA), and γ-tubulin (Sigma, St. Louis, MO).

    Techniques: Transgenic Assay, Mouse Assay, Staining

    Chromosome instability in eIF-5A2 transgenic mice . ( A ) Representative SKY images (left) and reversed DAPI stain (right) of bone marrow metaphase spreads from wild-type and transgenic mice. ( B, C ) Detection of hallmarks of chromosomal instability in eIF-5A2 transgenic MEFs, including misaligned chromosomes separated from the metaphase plate ( a' ), lagging chromosomes in anaphase ( b' and c' ), and micronuclei in interphase cells ( C ). ( D ) Quantification of the incidence of the micronuclei, misaligned and lagging chromosomes.

    Journal: BMC Cancer

    Article Title: Overexpression of eIF-5A2 in mice causes accelerated organismal aging by increasing chromosome instability

    doi: 10.1186/1471-2407-11-199

    Figure Lengend Snippet: Chromosome instability in eIF-5A2 transgenic mice . ( A ) Representative SKY images (left) and reversed DAPI stain (right) of bone marrow metaphase spreads from wild-type and transgenic mice. ( B, C ) Detection of hallmarks of chromosomal instability in eIF-5A2 transgenic MEFs, including misaligned chromosomes separated from the metaphase plate ( a' ), lagging chromosomes in anaphase ( b' and c' ), and micronuclei in interphase cells ( C ). ( D ) Quantification of the incidence of the micronuclei, misaligned and lagging chromosomes.

    Article Snippet: About 10 μg of lysate was separated by SDS-polyacrylamide gel electrophoresis, transferred to a PVDF Hybond-P membrane (Amersham Pharmacia Biotechnology, Piscataway, NJ), and detected by antibodies for eIF-5A2 (a mouse monoclonal antibody raised against the 54 residues of eIF-5A2), p53 (Zymed, San Francisco, CA), p21 (Upstate, Temecula, CA), p19 (Upstate, Lake Placid, NY), CDK4 (Cell Signaling Technology, Beverley, MA), and γ-tubulin (Sigma, St. Louis, MO).

    Techniques: Transgenic Assay, Mouse Assay, Staining

    Tissue distribution of transgenic eIF-5A2 protein and BrdU incorporation analysis of day 13.5 embryos . Expression of eIF-5A2 in developing liver, cartilage and neuroepithilium from transgenic and wild-type embryos (left) was detected by IHC (magnification, 400 ×). BrdU incorporation assay (right) was used to compare the proliferation rates between transgenic and wild-type embryos. No significant difference was observed in the percentage of BrdU positive cells between transgenic (56.3 ± 7.81) and wild-type embryos (52.73 ± 9.14, p > 0.05).

    Journal: BMC Cancer

    Article Title: Overexpression of eIF-5A2 in mice causes accelerated organismal aging by increasing chromosome instability

    doi: 10.1186/1471-2407-11-199

    Figure Lengend Snippet: Tissue distribution of transgenic eIF-5A2 protein and BrdU incorporation analysis of day 13.5 embryos . Expression of eIF-5A2 in developing liver, cartilage and neuroepithilium from transgenic and wild-type embryos (left) was detected by IHC (magnification, 400 ×). BrdU incorporation assay (right) was used to compare the proliferation rates between transgenic and wild-type embryos. No significant difference was observed in the percentage of BrdU positive cells between transgenic (56.3 ± 7.81) and wild-type embryos (52.73 ± 9.14, p > 0.05).

    Article Snippet: About 10 μg of lysate was separated by SDS-polyacrylamide gel electrophoresis, transferred to a PVDF Hybond-P membrane (Amersham Pharmacia Biotechnology, Piscataway, NJ), and detected by antibodies for eIF-5A2 (a mouse monoclonal antibody raised against the 54 residues of eIF-5A2), p53 (Zymed, San Francisco, CA), p21 (Upstate, Temecula, CA), p19 (Upstate, Lake Placid, NY), CDK4 (Cell Signaling Technology, Beverley, MA), and γ-tubulin (Sigma, St. Louis, MO).

    Techniques: Transgenic Assay, BrdU Incorporation Assay, Expressing, Immunohistochemistry

    eIF-5A2 expression, cell proliferation and histopathological analysis of 12-week-old mice (magnification, 400 ×). ( A ) Top panel, eIF-5A2 immunostaining of liver and heart from transgenic and wild-type mice. Middle panel, proliferation was measured by using PCNA immunostaining and the percentage of PCNA positive cells in transgenic mice (46.2 ± 10.98) was comparable to that in wild-type littermates (37.87 ± 10.44, p > 0.05). Bottom panel, haematoxylin and eosin (H E) staining of adult liver and heart tissues. ( B ) H E staining of the transgenic and wild-type livers after 12-week alcohol treatment.

    Journal: BMC Cancer

    Article Title: Overexpression of eIF-5A2 in mice causes accelerated organismal aging by increasing chromosome instability

    doi: 10.1186/1471-2407-11-199

    Figure Lengend Snippet: eIF-5A2 expression, cell proliferation and histopathological analysis of 12-week-old mice (magnification, 400 ×). ( A ) Top panel, eIF-5A2 immunostaining of liver and heart from transgenic and wild-type mice. Middle panel, proliferation was measured by using PCNA immunostaining and the percentage of PCNA positive cells in transgenic mice (46.2 ± 10.98) was comparable to that in wild-type littermates (37.87 ± 10.44, p > 0.05). Bottom panel, haematoxylin and eosin (H E) staining of adult liver and heart tissues. ( B ) H E staining of the transgenic and wild-type livers after 12-week alcohol treatment.

    Article Snippet: About 10 μg of lysate was separated by SDS-polyacrylamide gel electrophoresis, transferred to a PVDF Hybond-P membrane (Amersham Pharmacia Biotechnology, Piscataway, NJ), and detected by antibodies for eIF-5A2 (a mouse monoclonal antibody raised against the 54 residues of eIF-5A2), p53 (Zymed, San Francisco, CA), p21 (Upstate, Temecula, CA), p19 (Upstate, Lake Placid, NY), CDK4 (Cell Signaling Technology, Beverley, MA), and γ-tubulin (Sigma, St. Louis, MO).

    Techniques: Expressing, Mouse Assay, Immunostaining, Transgenic Assay, Staining

    Aging-related phenotypes in eIF5A2 transgenic mice . ( A ) Cumulative plot of body weight versus age of male eIF-5A2 transgenic mice (n = 11) and their wild-type siblings (n = 8). * P

    Journal: BMC Cancer

    Article Title: Overexpression of eIF-5A2 in mice causes accelerated organismal aging by increasing chromosome instability

    doi: 10.1186/1471-2407-11-199

    Figure Lengend Snippet: Aging-related phenotypes in eIF5A2 transgenic mice . ( A ) Cumulative plot of body weight versus age of male eIF-5A2 transgenic mice (n = 11) and their wild-type siblings (n = 8). * P

    Article Snippet: About 10 μg of lysate was separated by SDS-polyacrylamide gel electrophoresis, transferred to a PVDF Hybond-P membrane (Amersham Pharmacia Biotechnology, Piscataway, NJ), and detected by antibodies for eIF-5A2 (a mouse monoclonal antibody raised against the 54 residues of eIF-5A2), p53 (Zymed, San Francisco, CA), p21 (Upstate, Temecula, CA), p19 (Upstate, Lake Placid, NY), CDK4 (Cell Signaling Technology, Beverley, MA), and γ-tubulin (Sigma, St. Louis, MO).

    Techniques: Transgenic Assay, Mouse Assay

    Characterization of the eIF-5A2 transgenic MEF cells . ( A ) Senescence associated β-galactosidase staining in transgenic and wild-type MEFs. No difference was observed between these two MEFs. ( B ) Western blot analysis of lysates from transgenic and wild-type MEFs showed activation of eIF-5A2 repressed p19 level and therefore destabilized p53 and consequently repressed p21 in transgenic MEFs. ( C ) The replication capacity of both MEFs were measured by seeding 1 × 10 4 cells into one well of 6-well plate and counted every 3 days. Both wild-type and transgenic MEFs proliferated normally until passage 4 and proliferation capability declined during later passages (p5 and p6). ( D , E ) The comparison of cell growth curves of transgenic and wild-type MEFs in 10% serum ( D ) and 1% serum ( E ). The cell growth rate was significantly higher in transgenic MEFs than in wild-type MEFs ( p

    Journal: BMC Cancer

    Article Title: Overexpression of eIF-5A2 in mice causes accelerated organismal aging by increasing chromosome instability

    doi: 10.1186/1471-2407-11-199

    Figure Lengend Snippet: Characterization of the eIF-5A2 transgenic MEF cells . ( A ) Senescence associated β-galactosidase staining in transgenic and wild-type MEFs. No difference was observed between these two MEFs. ( B ) Western blot analysis of lysates from transgenic and wild-type MEFs showed activation of eIF-5A2 repressed p19 level and therefore destabilized p53 and consequently repressed p21 in transgenic MEFs. ( C ) The replication capacity of both MEFs were measured by seeding 1 × 10 4 cells into one well of 6-well plate and counted every 3 days. Both wild-type and transgenic MEFs proliferated normally until passage 4 and proliferation capability declined during later passages (p5 and p6). ( D , E ) The comparison of cell growth curves of transgenic and wild-type MEFs in 10% serum ( D ) and 1% serum ( E ). The cell growth rate was significantly higher in transgenic MEFs than in wild-type MEFs ( p

    Article Snippet: About 10 μg of lysate was separated by SDS-polyacrylamide gel electrophoresis, transferred to a PVDF Hybond-P membrane (Amersham Pharmacia Biotechnology, Piscataway, NJ), and detected by antibodies for eIF-5A2 (a mouse monoclonal antibody raised against the 54 residues of eIF-5A2), p53 (Zymed, San Francisco, CA), p21 (Upstate, Temecula, CA), p19 (Upstate, Lake Placid, NY), CDK4 (Cell Signaling Technology, Beverley, MA), and γ-tubulin (Sigma, St. Louis, MO).

    Techniques: Transgenic Assay, Staining, Western Blot, Activation Assay

    Generation of eIF-5A2 transgenic mice . ( A ) Three eIF-5A2 transgenic mouse founders (mouse No.: 10, 11, and 89) and their offspring (166-169) were determined by PCR. One mouse without eIF-5A2 transgene (No. 9) was used as negative control. Mouse fibroblast cell line NIH 3T3 and eIF-5A2 transgenic construct was used as negative (-) and positive (+) controls. ( B ) The transgene eIF-5A2 was mapped to one mouse chromosome site in line 11 by FISH. The metaphase spread was prepared from bone marrow lymphocyte. Arrow indicates the hybridization signals of eIF-5A2 . ( C ) Expression of eIF-5A2 in transgenic mice was confirmed by Northern blot analysis. A human eIF-5A2 cDNA probe was used and it did not detect the endogenous eIF-5A2 mRNA in both wild-type and transgenic mice. ( D ) Western blot showed the overexpression of human eIF-5A2 in transgenic mice using liver lysates. The lower bands (17dD) were eIF-5A2 and the upper bands were shifted eIF-5A2 bands caused by posttranslational hypusination. ( E ) Expression of transgene eIF-5A2 in various tissues of transgenic mouse was detected by RT-PCR using a pair of human-specific primers.

    Journal: BMC Cancer

    Article Title: Overexpression of eIF-5A2 in mice causes accelerated organismal aging by increasing chromosome instability

    doi: 10.1186/1471-2407-11-199

    Figure Lengend Snippet: Generation of eIF-5A2 transgenic mice . ( A ) Three eIF-5A2 transgenic mouse founders (mouse No.: 10, 11, and 89) and their offspring (166-169) were determined by PCR. One mouse without eIF-5A2 transgene (No. 9) was used as negative control. Mouse fibroblast cell line NIH 3T3 and eIF-5A2 transgenic construct was used as negative (-) and positive (+) controls. ( B ) The transgene eIF-5A2 was mapped to one mouse chromosome site in line 11 by FISH. The metaphase spread was prepared from bone marrow lymphocyte. Arrow indicates the hybridization signals of eIF-5A2 . ( C ) Expression of eIF-5A2 in transgenic mice was confirmed by Northern blot analysis. A human eIF-5A2 cDNA probe was used and it did not detect the endogenous eIF-5A2 mRNA in both wild-type and transgenic mice. ( D ) Western blot showed the overexpression of human eIF-5A2 in transgenic mice using liver lysates. The lower bands (17dD) were eIF-5A2 and the upper bands were shifted eIF-5A2 bands caused by posttranslational hypusination. ( E ) Expression of transgene eIF-5A2 in various tissues of transgenic mouse was detected by RT-PCR using a pair of human-specific primers.

    Article Snippet: About 10 μg of lysate was separated by SDS-polyacrylamide gel electrophoresis, transferred to a PVDF Hybond-P membrane (Amersham Pharmacia Biotechnology, Piscataway, NJ), and detected by antibodies for eIF-5A2 (a mouse monoclonal antibody raised against the 54 residues of eIF-5A2), p53 (Zymed, San Francisco, CA), p21 (Upstate, Temecula, CA), p19 (Upstate, Lake Placid, NY), CDK4 (Cell Signaling Technology, Beverley, MA), and γ-tubulin (Sigma, St. Louis, MO).

    Techniques: Transgenic Assay, Mouse Assay, Polymerase Chain Reaction, Negative Control, Construct, Fluorescence In Situ Hybridization, Hybridization, Expressing, Northern Blot, Western Blot, Over Expression, Reverse Transcription Polymerase Chain Reaction