flag tagged trkb  (Millipore)


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    Structured Review

    Millipore flag tagged trkb
    Trk receptors are transcytosed from neuronal soma surfaces to axons. (A) Schematic of antibody feeding assay in compartmentalized cultures expressing <t>FLAG-TrkB:A</t> to follow transcytosis of Trk receptors originating from neuronal soma surfaces. (B) Representative
    Flag Tagged Trkb, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 2867 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 85 stars, based on 2867 article reviews
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    flag tagged trkb - by Bioz Stars, 2020-09
    85/100 stars

    Images

    1) Product Images from "Axonal Targeting of Trk Receptors via Transcytosis Regulates Sensitivity to Neurotrophin Responses"

    Article Title: Axonal Targeting of Trk Receptors via Transcytosis Regulates Sensitivity to Neurotrophin Responses

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    doi: 10.1523/JNEUROSCI.1542-09.2009

    Trk receptors are transcytosed from neuronal soma surfaces to axons. (A) Schematic of antibody feeding assay in compartmentalized cultures expressing FLAG-TrkB:A to follow transcytosis of Trk receptors originating from neuronal soma surfaces. (B) Representative
    Figure Legend Snippet: Trk receptors are transcytosed from neuronal soma surfaces to axons. (A) Schematic of antibody feeding assay in compartmentalized cultures expressing FLAG-TrkB:A to follow transcytosis of Trk receptors originating from neuronal soma surfaces. (B) Representative

    Techniques Used: Feeding Assay, Expressing

    Rab11a mediates Trk transcytosis in sympathetic neurons. (A) FLAG-TrkB:A receptors undergoing transcytosis co-localize with Rab11a in axons (arrowheads) as detected by immunostaining for FLAG antibody and endogenous Rab11a. Antibody-feeding assay was
    Figure Legend Snippet: Rab11a mediates Trk transcytosis in sympathetic neurons. (A) FLAG-TrkB:A receptors undergoing transcytosis co-localize with Rab11a in axons (arrowheads) as detected by immunostaining for FLAG antibody and endogenous Rab11a. Antibody-feeding assay was

    Techniques Used: Immunostaining, Feeding Assay

    2) Product Images from "Serodiversity of Opsonic Antibodies against Enterococcus faecalis --Glycans of the Cell Wall Revisited"

    Article Title: Serodiversity of Opsonic Antibodies against Enterococcus faecalis --Glycans of the Cell Wall Revisited

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0017839

    Relationship of diheteroglycan to the biosynthetic product of the cps locus. ( A ) Loss of opsonic activity after absorption of rabbit antiserum raised against E. faecalis type 2 (CPS-C) diheteroglycan with whole bacteria. Before the assay the serum was absorbed with either E. faecalis FA2-2 (CPS-C) wild type or its isogenic, acapsular cpsI mutant ( E. faecalis HG101) for 60 min. Absorbed serum was used at a final dilution of 1∶40 in the assay. Bars represent the mean of four determinations and the error bar the SEM. ( B ) Native PAGE of purified diheteroglycan from E. faecalis type 2 (lane 1), cell wall lysates of E. faecalis FA2-2 wild type (CPS-C, lane 2) and the isogenic cpsI mutant (HG101, lane 3). Cell envelope carbohydrates were released by digestion of peptidoglycan by lysozyme and mutanolysin and acrylamide gels were stained with Stains-All according to the method of Hancock et al. [12] .
    Figure Legend Snippet: Relationship of diheteroglycan to the biosynthetic product of the cps locus. ( A ) Loss of opsonic activity after absorption of rabbit antiserum raised against E. faecalis type 2 (CPS-C) diheteroglycan with whole bacteria. Before the assay the serum was absorbed with either E. faecalis FA2-2 (CPS-C) wild type or its isogenic, acapsular cpsI mutant ( E. faecalis HG101) for 60 min. Absorbed serum was used at a final dilution of 1∶40 in the assay. Bars represent the mean of four determinations and the error bar the SEM. ( B ) Native PAGE of purified diheteroglycan from E. faecalis type 2 (lane 1), cell wall lysates of E. faecalis FA2-2 wild type (CPS-C, lane 2) and the isogenic cpsI mutant (HG101, lane 3). Cell envelope carbohydrates were released by digestion of peptidoglycan by lysozyme and mutanolysin and acrylamide gels were stained with Stains-All according to the method of Hancock et al. [12] .

    Techniques Used: Activity Assay, Mutagenesis, Clear Native PAGE, Purification, Staining

    3) Product Images from "Hydrogen Sulfide Improves Cardiomyocyte Function in a Cardiac Arrest Model"

    Article Title: Hydrogen Sulfide Improves Cardiomyocyte Function in a Cardiac Arrest Model

    Journal: Annals of Transplantation

    doi: 10.12659/AOT.901410

    Effect of GYY4137 on ATP content and protein synthesis in HL-1 cells. ( A ) Relative ATP content during preconditioning. ATP content is represented as relative content to non-preconditioned cells (0 hour). Dashed line represents cells treated with active GYY4137 and solid line represents cells treated with the inactive GYY4137. ( B ) Top panel , dot blot assay showing methionine L-azidohomoalanine (AHA) incorporation at 3 hours of preconditioning. Bottom panel , experimental set up for AHA incorporation: Negative control, non-biotinylated AHA; positive control, exponential cell growth in complete Claycomb medium; no analogue, biotinylation of extract without AHA incorporation; actidione, exponential cell growth in complete Claycomb medium after pre-treatment with the protein synthesis inhibitor cycloheximide. ( C ) Relative ATP content during nutrient starvation. ATP content is represented as relative content to the initial level of control cells (0 hour). Dashed line represents cells treated with active GYY4137 and solid line represents cells treated with inactive GYY4137. ( D ) Dot blot assay showing AHA incorporation at 30 minutes (0.5 hours), 1 hour, and 3 hours after starvation. * p
    Figure Legend Snippet: Effect of GYY4137 on ATP content and protein synthesis in HL-1 cells. ( A ) Relative ATP content during preconditioning. ATP content is represented as relative content to non-preconditioned cells (0 hour). Dashed line represents cells treated with active GYY4137 and solid line represents cells treated with the inactive GYY4137. ( B ) Top panel , dot blot assay showing methionine L-azidohomoalanine (AHA) incorporation at 3 hours of preconditioning. Bottom panel , experimental set up for AHA incorporation: Negative control, non-biotinylated AHA; positive control, exponential cell growth in complete Claycomb medium; no analogue, biotinylation of extract without AHA incorporation; actidione, exponential cell growth in complete Claycomb medium after pre-treatment with the protein synthesis inhibitor cycloheximide. ( C ) Relative ATP content during nutrient starvation. ATP content is represented as relative content to the initial level of control cells (0 hour). Dashed line represents cells treated with active GYY4137 and solid line represents cells treated with inactive GYY4137. ( D ) Dot blot assay showing AHA incorporation at 30 minutes (0.5 hours), 1 hour, and 3 hours after starvation. * p

    Techniques Used: Dot Blot, Negative Control, Positive Control

    GYY4137 improves cardioplegic cardio-protective performance during cardiac arrest. ( A ) Scheme of the procedure to test cardioplegic solutions in rat hearts utilizing a Langendorff apparatus. ( B ) Western blotting for caspase-3 in rat heart tissues perfused with Conv or del Nido cardioplegic solutions with or without GYY4137 (n=4). The graph shows quantification of densitometry analysis utilizing ImageJ software. ( C ) ATP quantification in rat heart tissue after finalization of Langendorff perfusion protocol as described in methods section. For the sham group, rat hearts were not subjected to the experimental Langendorff protocol; the organs were directly explanted and ATP content was analyzed. * p
    Figure Legend Snippet: GYY4137 improves cardioplegic cardio-protective performance during cardiac arrest. ( A ) Scheme of the procedure to test cardioplegic solutions in rat hearts utilizing a Langendorff apparatus. ( B ) Western blotting for caspase-3 in rat heart tissues perfused with Conv or del Nido cardioplegic solutions with or without GYY4137 (n=4). The graph shows quantification of densitometry analysis utilizing ImageJ software. ( C ) ATP quantification in rat heart tissue after finalization of Langendorff perfusion protocol as described in methods section. For the sham group, rat hearts were not subjected to the experimental Langendorff protocol; the organs were directly explanted and ATP content was analyzed. * p

    Techniques Used: Western Blot, Software

    Effect of GYY4137 on HL-1 cell apoptosis. ( A ) Effect of GYY4137 preconditioning on apoptosis of cells cultured in glucose and amino acid deprivation medium (-Glu KH). ( B ) Effect of GYY4137 preconditioning on apoptosis of cells cultured in amino acid deprivation medium (KH). ( C, D ) Effect of GYY4137 preconditioning on apoptosis of cells treated with staurosporine ( C ) and doxorubicin ( D ). ( E ) Effect of GYY4137 on apoptosis of cells during starvation in -Glu KH medium without preconditioning. In all cases, results show percentage of apoptosis over time. Dashed lines represent cells treated with active GYY4137 and solid lines represent cells treated with inactive GYY4137. Three replicates in three differences experiments were performed per condition. * p
    Figure Legend Snippet: Effect of GYY4137 on HL-1 cell apoptosis. ( A ) Effect of GYY4137 preconditioning on apoptosis of cells cultured in glucose and amino acid deprivation medium (-Glu KH). ( B ) Effect of GYY4137 preconditioning on apoptosis of cells cultured in amino acid deprivation medium (KH). ( C, D ) Effect of GYY4137 preconditioning on apoptosis of cells treated with staurosporine ( C ) and doxorubicin ( D ). ( E ) Effect of GYY4137 on apoptosis of cells during starvation in -Glu KH medium without preconditioning. In all cases, results show percentage of apoptosis over time. Dashed lines represent cells treated with active GYY4137 and solid lines represent cells treated with inactive GYY4137. Three replicates in three differences experiments were performed per condition. * p

    Techniques Used: Cell Culture

    Short-term electrical recovery after GYY4137 treatment in the isolated Langendorff-perfused rat model. ( A ) Representative 5 second volume-conducted pseudo-ECG traces showing pre-treatment (Pre, light) or post-treatment (Post, dark) with cardioplegic solutions. A better preservation of heart rate (HR) and more regular cardiac activation is detected with del Nido+GYY4137 formulation than with untreated KH solution-preserved hearts. Traces are segmented after 15 minutes of registration from a single-lead electrode located at the epicardial base of the right ventricle in the Langendorff-perfused whole-heart. ( B ) Quantification of the effects of cardioplegic treatment on heart rate (HR, beats per minute) reveals significant slowing of cardiac activation, whereas heartrate-slowing is non-significantly different in the del Nido + GYY4137 treated group. * p
    Figure Legend Snippet: Short-term electrical recovery after GYY4137 treatment in the isolated Langendorff-perfused rat model. ( A ) Representative 5 second volume-conducted pseudo-ECG traces showing pre-treatment (Pre, light) or post-treatment (Post, dark) with cardioplegic solutions. A better preservation of heart rate (HR) and more regular cardiac activation is detected with del Nido+GYY4137 formulation than with untreated KH solution-preserved hearts. Traces are segmented after 15 minutes of registration from a single-lead electrode located at the epicardial base of the right ventricle in the Langendorff-perfused whole-heart. ( B ) Quantification of the effects of cardioplegic treatment on heart rate (HR, beats per minute) reveals significant slowing of cardiac activation, whereas heartrate-slowing is non-significantly different in the del Nido + GYY4137 treated group. * p

    Techniques Used: Isolation, Preserving, Activation Assay

    GYY4137 reduces oxidative stress during cardiac arrest. ( A ) Levels of oxidative stress biomarkers GSH, GSSG; and ( B ) SAM and SAH were analyzed in rat hearts treated with Conv or del Nido cardioplegia solutions with or without GYY4137. Graphs show the ratio of GSH/GSSG ( A ) and SAM/SAH ( B ). Four-six experiments were performed per condition. * p
    Figure Legend Snippet: GYY4137 reduces oxidative stress during cardiac arrest. ( A ) Levels of oxidative stress biomarkers GSH, GSSG; and ( B ) SAM and SAH were analyzed in rat hearts treated with Conv or del Nido cardioplegia solutions with or without GYY4137. Graphs show the ratio of GSH/GSSG ( A ) and SAM/SAH ( B ). Four-six experiments were performed per condition. * p

    Techniques Used:

    4) Product Images from "The proline rich domain of p53 is dispensable for MGMT-dependent DNA repair and cell survival following alkylation damage"

    Article Title: The proline rich domain of p53 is dispensable for MGMT-dependent DNA repair and cell survival following alkylation damage

    Journal: Cell Death and Differentiation

    doi: 10.1038/cdd.2017.116

    MGMT overexpression facilitates repair and some survival advantage in p53 −/− cells. ( a ) Cell death at 18 h as measured by flow cytometric analysis of Annexin V staining in MEF cells overexpressing MGMT and treated with 100 μ M Diazald. ( b ) Intracellular pH2AX levels measured by flow cytometry in MEF cells overexpressing MGMT and treated with 100 μ M Diazald for 3 h to assess damage, or at 24 h to assess repair. ( c ) Colony-forming assays examining survival in p53 WT , p53 ΔP or p53 −/− MEF cells with or without MGMT overexpression after 250 μ M Diazald for increasing time intervals. ( d ) Quantification of colony-forming assay in p53 WT , p53 ΔP , or p53 −/− MEF cells
    Figure Legend Snippet: MGMT overexpression facilitates repair and some survival advantage in p53 −/− cells. ( a ) Cell death at 18 h as measured by flow cytometric analysis of Annexin V staining in MEF cells overexpressing MGMT and treated with 100 μ M Diazald. ( b ) Intracellular pH2AX levels measured by flow cytometry in MEF cells overexpressing MGMT and treated with 100 μ M Diazald for 3 h to assess damage, or at 24 h to assess repair. ( c ) Colony-forming assays examining survival in p53 WT , p53 ΔP or p53 −/− MEF cells with or without MGMT overexpression after 250 μ M Diazald for increasing time intervals. ( d ) Quantification of colony-forming assay in p53 WT , p53 ΔP , or p53 −/− MEF cells

    Techniques Used: Over Expression, Flow Cytometry, Staining, Cytometry

    The repair gene MGMT is induced in p53 ΔP cells upon alkylating damage and mediates DNA repair and cell survival in cells. ( a ) MGMT expression levels 6–8 h IR (5 Gy) in irradiated immune cells ( in vitro ) or immune cells isolated from irradiated mice ( ex vivo ). MGMT expression levels in thymocytes treated with 50 μ M Diazald for 18 h. ( b ) Intracellular pH2AX levels measured by flow cytometry in p53 WT , p53 ΔP and p53 −/− MEF cell treated with 100 μ M Diazald at 3 h to assess damage, or at 24 h to assess repair
    Figure Legend Snippet: The repair gene MGMT is induced in p53 ΔP cells upon alkylating damage and mediates DNA repair and cell survival in cells. ( a ) MGMT expression levels 6–8 h IR (5 Gy) in irradiated immune cells ( in vitro ) or immune cells isolated from irradiated mice ( ex vivo ). MGMT expression levels in thymocytes treated with 50 μ M Diazald for 18 h. ( b ) Intracellular pH2AX levels measured by flow cytometry in p53 WT , p53 ΔP and p53 −/− MEF cell treated with 100 μ M Diazald at 3 h to assess damage, or at 24 h to assess repair

    Techniques Used: Expressing, Irradiation, In Vitro, Isolation, Mouse Assay, Ex Vivo, Flow Cytometry, Cytometry

    Loss of MGMT in cells inhibits repair and survival of p53 ΔP cells. Intracellular pH2AX levels measured by flow cytometry in p53 WT , p53 ΔP and p53 −/− MEF cells ( a ) sufficient or deficient for MGMT or, ( d ) expressing BCLxL, treated with 100 μ M Diazald for 3 h to assess damage or 24 h to assess repair. ( b ) Cell death as measured flow cytometric analysis of Annexin V staining in MEF cells treated for 3 h with 100 μ M Diazald, washed and assessed for Annexin V staining 24 h later. ( c ) Cell death as measured flow cytometric analysis of Annexin V staining in MEF cells expressing BCLxL and treated with 100 μ M Diazald for 24 h. ( e ) Colony-forming assays examining survival in Left : p53 WT , p53 ΔP and p53 −/− MEF cells sufficient or deficient for MGMT; Right : MEF cells from p53 ΔP , MGMT −/− mice after 250 μ M Diazald treatment for increasing time intervals
    Figure Legend Snippet: Loss of MGMT in cells inhibits repair and survival of p53 ΔP cells. Intracellular pH2AX levels measured by flow cytometry in p53 WT , p53 ΔP and p53 −/− MEF cells ( a ) sufficient or deficient for MGMT or, ( d ) expressing BCLxL, treated with 100 μ M Diazald for 3 h to assess damage or 24 h to assess repair. ( b ) Cell death as measured flow cytometric analysis of Annexin V staining in MEF cells treated for 3 h with 100 μ M Diazald, washed and assessed for Annexin V staining 24 h later. ( c ) Cell death as measured flow cytometric analysis of Annexin V staining in MEF cells expressing BCLxL and treated with 100 μ M Diazald for 24 h. ( e ) Colony-forming assays examining survival in Left : p53 WT , p53 ΔP and p53 −/− MEF cells sufficient or deficient for MGMT; Right : MEF cells from p53 ΔP , MGMT −/− mice after 250 μ M Diazald treatment for increasing time intervals

    Techniques Used: Flow Cytometry, Cytometry, Expressing, Staining, Mouse Assay

    5) Product Images from "Expression of stage-specific embryonic antigen-4 (SSEA-4) defines spontaneous loss of epithelial phenotype in human solid tumor cells"

    Article Title: Expression of stage-specific embryonic antigen-4 (SSEA-4) defines spontaneous loss of epithelial phenotype in human solid tumor cells

    Journal: Glycobiology

    doi: 10.1093/glycob/cwv032

    ST3GAL2 knockdown impairs SSEA-4 expression and cell adhesion. ( A and B ) Modulation of ST3GAL2 mRNA in DU145 cells by lentiviral ST3GAL2 knockdown impairs SSEA-4 expression. Panel (A) shows relative gene expression levels of ST3GAL2 normalized to internal
    Figure Legend Snippet: ST3GAL2 knockdown impairs SSEA-4 expression and cell adhesion. ( A and B ) Modulation of ST3GAL2 mRNA in DU145 cells by lentiviral ST3GAL2 knockdown impairs SSEA-4 expression. Panel (A) shows relative gene expression levels of ST3GAL2 normalized to internal

    Techniques Used: Expressing

    6) Product Images from "A new method to evaluate anti-allergic effect of food component by measuring leukotriene B4 from a mouse mast cell line"

    Article Title: A new method to evaluate anti-allergic effect of food component by measuring leukotriene B4 from a mouse mast cell line

    Journal: Cytotechnology

    doi: 10.1007/s10616-017-0129-9

    Effect of soybean isoflavones and equol on LTB 4 production by PB-3c. PB-3c was pre-cultured with 50 µM AA for 48 h and then stimulated with 5 µM A23187 in the presence of 50 µM daidzin, genistin, daidzein, genistein, and equol. LTB 4 was determined by HPLC with UV detection. Data represent mean ± SE (n = 4). Means without a common letter are significantly different ( p
    Figure Legend Snippet: Effect of soybean isoflavones and equol on LTB 4 production by PB-3c. PB-3c was pre-cultured with 50 µM AA for 48 h and then stimulated with 5 µM A23187 in the presence of 50 µM daidzin, genistin, daidzein, genistein, and equol. LTB 4 was determined by HPLC with UV detection. Data represent mean ± SE (n = 4). Means without a common letter are significantly different ( p

    Techniques Used: Cell Culture, High Performance Liquid Chromatography

    Effect of flavonoids on LTB 4 production by PB-3c. PB-3c was pre-cultured with 50 µM AA for 48 h and then stimulated with 5 µM A23187 in the presence of 50 µM quercetin and kaempferol. LTB 4 was determined by HPLC with UV detection. Data represent mean ± SE (n = 4). Means without a common letter are significantly different ( p
    Figure Legend Snippet: Effect of flavonoids on LTB 4 production by PB-3c. PB-3c was pre-cultured with 50 µM AA for 48 h and then stimulated with 5 µM A23187 in the presence of 50 µM quercetin and kaempferol. LTB 4 was determined by HPLC with UV detection. Data represent mean ± SE (n = 4). Means without a common letter are significantly different ( p

    Techniques Used: Cell Culture, High Performance Liquid Chromatography

    Effect of arachidonic acid (AA) concentration during pre-culture of PB-3c on LTB 4 production. A mast cell line, PB-3c (2 × 10 6 cells) was pre-cultured in RPMI-1640 containing 0–100 µM of AA for 48 h. The cells were stimulated with 5 µM calcium ionophore (A23187) for 20 min and then LTB 4 produced by the cells was determined by HPLC with UV detection. Data represent mean ± SE (n = 4). Means without a common letter are significantly different ( p
    Figure Legend Snippet: Effect of arachidonic acid (AA) concentration during pre-culture of PB-3c on LTB 4 production. A mast cell line, PB-3c (2 × 10 6 cells) was pre-cultured in RPMI-1640 containing 0–100 µM of AA for 48 h. The cells were stimulated with 5 µM calcium ionophore (A23187) for 20 min and then LTB 4 produced by the cells was determined by HPLC with UV detection. Data represent mean ± SE (n = 4). Means without a common letter are significantly different ( p

    Techniques Used: Concentration Assay, Cell Culture, Produced, High Performance Liquid Chromatography

    Effect of enzyme inhibitors related to arachidonate cascade on LTB 4 production by PB-3c. PB-3c was pre-cultured with 50 µM AA for 48 h and then stimulated with 5 µM A23187 in the presence of MK-886 (5-LOX inhibitor) and AACOCF 3 (phospholipase A 2 inhibitor). LTB 4 was determined by HPLC with UV detection. Data represent mean ± SE (n = 4). Means without a common letter are significantly different ( p
    Figure Legend Snippet: Effect of enzyme inhibitors related to arachidonate cascade on LTB 4 production by PB-3c. PB-3c was pre-cultured with 50 µM AA for 48 h and then stimulated with 5 µM A23187 in the presence of MK-886 (5-LOX inhibitor) and AACOCF 3 (phospholipase A 2 inhibitor). LTB 4 was determined by HPLC with UV detection. Data represent mean ± SE (n = 4). Means without a common letter are significantly different ( p

    Techniques Used: Cell Culture, High Performance Liquid Chromatography

    Effect of pre-culture period of PB-3c with AA before the stimulation on LTB 4 production. PB-3c (2 × 10 6 cells) was pre-cultured in RPMI-1640 containing 50 µM AA for 0–72 h. The cells were stimulated with 5 µM A23187 for 20 min and then LTB 4 produced by the cells was determined by HPLC with UV detection. Data represent mean ± SE (n = 4)
    Figure Legend Snippet: Effect of pre-culture period of PB-3c with AA before the stimulation on LTB 4 production. PB-3c (2 × 10 6 cells) was pre-cultured in RPMI-1640 containing 50 µM AA for 0–72 h. The cells were stimulated with 5 µM A23187 for 20 min and then LTB 4 produced by the cells was determined by HPLC with UV detection. Data represent mean ± SE (n = 4)

    Techniques Used: Cell Culture, Produced, High Performance Liquid Chromatography

    7) Product Images from "Contributions to Protection from Streptococcus pneumoniae Infection Using the Monovalent Recombinant Protein Vaccine Candidates PcpA, PhtD, and PlyD1 in an Infant Murine Model during Challenge"

    Article Title: Contributions to Protection from Streptococcus pneumoniae Infection Using the Monovalent Recombinant Protein Vaccine Candidates PcpA, PhtD, and PlyD1 in an Infant Murine Model during Challenge

    Journal: Clinical and Vaccine Immunology : CVI

    doi: 10.1128/CVI.00052-14

    Total specific and isotype antibody titers elicited with monovalent vaccines. Infant and adult mice were vaccinated using monovalent formulations of PcpA (0.2 μg), PhtD (0.9 μg), or PlyD1 (5 μg). (A) Total antigen-specific antibody
    Figure Legend Snippet: Total specific and isotype antibody titers elicited with monovalent vaccines. Infant and adult mice were vaccinated using monovalent formulations of PcpA (0.2 μg), PhtD (0.9 μg), or PlyD1 (5 μg). (A) Total antigen-specific antibody

    Techniques Used: Mouse Assay

    Vaccination with monovalent antigens shows enhanced bacterial clearance. (A) We assessed bacterial lung burdens in PcpA-vaccinated (A), PhtD-vaccinated (B), or PlyD1-vaccinated (C) infant and adult mice. n = 5 for 3 independent experiments; means and
    Figure Legend Snippet: Vaccination with monovalent antigens shows enhanced bacterial clearance. (A) We assessed bacterial lung burdens in PcpA-vaccinated (A), PhtD-vaccinated (B), or PlyD1-vaccinated (C) infant and adult mice. n = 5 for 3 independent experiments; means and

    Techniques Used: Mouse Assay

    Infant mice were injected at the times shown with monovalent vaccines containing PcpA, PhtD, or PlyD1. Injections were into both hind leg caudal muscles (25 μl per muscle). Four weeks (wks) after the third vaccination, the mice were challenged
    Figure Legend Snippet: Infant mice were injected at the times shown with monovalent vaccines containing PcpA, PhtD, or PlyD1. Injections were into both hind leg caudal muscles (25 μl per muscle). Four weeks (wks) after the third vaccination, the mice were challenged

    Techniques Used: Mouse Assay, Injection

    Vaccination leads to a reduction in S. pneumoniae adherence to primary lung epithelial cells. (A to F) FITC-labeled S. pneumoniae was incubated with serum from vaccinated (PcpA or PhtD monovalent or mixed antibodies) or unvaccinated mice and with guinea
    Figure Legend Snippet: Vaccination leads to a reduction in S. pneumoniae adherence to primary lung epithelial cells. (A to F) FITC-labeled S. pneumoniae was incubated with serum from vaccinated (PcpA or PhtD monovalent or mixed antibodies) or unvaccinated mice and with guinea

    Techniques Used: Labeling, Incubation, Mouse Assay

    8) Product Images from "DNA Excision Repair and DNA Damage-Induced Apoptosis Are Linked to Poly(ADP-Ribosyl)ation but Have Different Requirements for p53"

    Article Title: DNA Excision Repair and DNA Damage-Induced Apoptosis Are Linked to Poly(ADP-Ribosyl)ation but Have Different Requirements for p53

    Journal: Molecular and Cellular Biology

    doi:

    Expression levels of PARP, the PARP DBD transgene, p53, and Bax upon X irradiation in transgenic, null, and combinatorial mutant mice. The immunoblot shows expression levels of the proteins in thymocytes from the indicated mice 3 h after X irradiation (2 Gy). Equal loading of each lane was controlled by Ponceau staining (not shown).
    Figure Legend Snippet: Expression levels of PARP, the PARP DBD transgene, p53, and Bax upon X irradiation in transgenic, null, and combinatorial mutant mice. The immunoblot shows expression levels of the proteins in thymocytes from the indicated mice 3 h after X irradiation (2 Gy). Equal loading of each lane was controlled by Ponceau staining (not shown).

    Techniques Used: Expressing, Irradiation, Transgenic Assay, Mutagenesis, Mouse Assay, Staining

    High levels of Bcl-2 block sensitization of thymocytes for apoptosis by PARP DBD but do not interfere with DNA repair. (a) Thymocytes were explanted from lck -PARP DBD, Eμ bcl-2 , or double-transgenic mice and from nontransgenic (non tg) controls and were treated with MNNG (40 μM). Before treatment and at different times thereafter, samples were analyzed for the percentage of apoptotic cells. Shown are mean values with standard deviations representative of three independent sets of experiments. (b) Comet assay to compare DNA repair kinetics after MNNG treatment between cells from lck -PARP DBD mice, Eμ bcl-2 animals, and mice carry both an lck -PARP DBD transgene and an Eμ bcl-2 transgene (cells from age-matched nontransgenic littermate controls behaved like cells from Eμ bcl-2 mice [not shown]). Given are average values with standard deviations from 40 cells per sample and time point against elapsed time after the stimulus. In each case, a representative of three independent experiments is depicted.
    Figure Legend Snippet: High levels of Bcl-2 block sensitization of thymocytes for apoptosis by PARP DBD but do not interfere with DNA repair. (a) Thymocytes were explanted from lck -PARP DBD, Eμ bcl-2 , or double-transgenic mice and from nontransgenic (non tg) controls and were treated with MNNG (40 μM). Before treatment and at different times thereafter, samples were analyzed for the percentage of apoptotic cells. Shown are mean values with standard deviations representative of three independent sets of experiments. (b) Comet assay to compare DNA repair kinetics after MNNG treatment between cells from lck -PARP DBD mice, Eμ bcl-2 animals, and mice carry both an lck -PARP DBD transgene and an Eμ bcl-2 transgene (cells from age-matched nontransgenic littermate controls behaved like cells from Eμ bcl-2 mice [not shown]). Given are average values with standard deviations from 40 cells per sample and time point against elapsed time after the stimulus. In each case, a representative of three independent experiments is depicted.

    Techniques Used: Blocking Assay, Transgenic Assay, Mouse Assay, Single Cell Gel Electrophoresis

    Influence of p53 and PARP deficiency on DNA resealing after induction of damage by X irradiation. (a) Comet assay to compare DNA repair kinetics after X irradiation with a single dose of 2 Gy between cells from nontransgenic (non tg) controls, PARP null mice (PARP −/− ), lck -PARP DBD mice, and mutant mice that both lack the PARP gene and express the PARP DBD transgene (lck-PARP DBD, PARP −/− ). (b) Comparison of DNA repair kinetics from PARP null mice (PARP −/− ), p53-deficient animals (p53 −/− ), mice that are both p53 deficient and PARP null (p53 −/− , PARP −/− ), and age-matched nontransgenic littermate controls. Given are average values with standard deviations from 40 cells per sample and time point against elapsed time after the stimulus. In each case, a representative of three independent experiments is depicted. For measurement of the comets, a fluorescence microscope was coupled with an intensified target camera, and an interactive digital image analysis system was established.
    Figure Legend Snippet: Influence of p53 and PARP deficiency on DNA resealing after induction of damage by X irradiation. (a) Comet assay to compare DNA repair kinetics after X irradiation with a single dose of 2 Gy between cells from nontransgenic (non tg) controls, PARP null mice (PARP −/− ), lck -PARP DBD mice, and mutant mice that both lack the PARP gene and express the PARP DBD transgene (lck-PARP DBD, PARP −/− ). (b) Comparison of DNA repair kinetics from PARP null mice (PARP −/− ), p53-deficient animals (p53 −/− ), mice that are both p53 deficient and PARP null (p53 −/− , PARP −/− ), and age-matched nontransgenic littermate controls. Given are average values with standard deviations from 40 cells per sample and time point against elapsed time after the stimulus. In each case, a representative of three independent experiments is depicted. For measurement of the comets, a fluorescence microscope was coupled with an intensified target camera, and an interactive digital image analysis system was established.

    Techniques Used: Irradiation, Single Cell Gel Electrophoresis, Mouse Assay, Mutagenesis, Fluorescence, Microscopy

    Introduction of a functionally active dominant negative mutant of PARP into the germ line of transgenic mice. (a) Schematic representation of the construct used to express the DBD of PARP in T cells of transgenic mice. (b) Expression of the lck -PARP DBD transgene in extracts from thymi of mice from four different transgenic lines ( lck -PARP DBD lines 1, 2, 3, and 4). The level of transgene expression was measured by direct scanning of the ECL-treated membrane after blotting with a video camera and subsequent analysis by the AIDA software (Raytest, Straubenhardt, Germany) on a Fuji phosphorimager. PARP DBD expression was found to be higher in lines 1 and 2 than in lines 3 and 4. The PARP DBD protein was detected with the monoclonal antibody CII10, which recognizes the hPARP transgene and the endogenous mPARP. Relative expression levels are given in arbitrary units (au) obtained through the scanning procedure. (c) Dominant negative effect of the expression of the PARP DBD in T cells. Thymocytes from wild-type and lck -PARP DBD transgenic mice were treated with MNNG, and the formation of poly(ADP-ribose) was followed by immunofluorescence. Compared to nontransgenic (non tg) controls, thymocytes from lck -PARP DBD transgenic mice lack detectable polymer formation after MNNG treatment. (d) Expression level of the PARP DBD in different organs of a transgenic mouse of lck -PARP DBD line 2. As a control, an extract from a nontransgenic thymus was loaded. The PARP DBD protein was detected with the monoclonal antibody CII10, which recognizes the hPARP transgene and the endogenous mPARP. The relative amount of PARP DBD protein expression in splenocytes is detectable but very low (data not shown).
    Figure Legend Snippet: Introduction of a functionally active dominant negative mutant of PARP into the germ line of transgenic mice. (a) Schematic representation of the construct used to express the DBD of PARP in T cells of transgenic mice. (b) Expression of the lck -PARP DBD transgene in extracts from thymi of mice from four different transgenic lines ( lck -PARP DBD lines 1, 2, 3, and 4). The level of transgene expression was measured by direct scanning of the ECL-treated membrane after blotting with a video camera and subsequent analysis by the AIDA software (Raytest, Straubenhardt, Germany) on a Fuji phosphorimager. PARP DBD expression was found to be higher in lines 1 and 2 than in lines 3 and 4. The PARP DBD protein was detected with the monoclonal antibody CII10, which recognizes the hPARP transgene and the endogenous mPARP. Relative expression levels are given in arbitrary units (au) obtained through the scanning procedure. (c) Dominant negative effect of the expression of the PARP DBD in T cells. Thymocytes from wild-type and lck -PARP DBD transgenic mice were treated with MNNG, and the formation of poly(ADP-ribose) was followed by immunofluorescence. Compared to nontransgenic (non tg) controls, thymocytes from lck -PARP DBD transgenic mice lack detectable polymer formation after MNNG treatment. (d) Expression level of the PARP DBD in different organs of a transgenic mouse of lck -PARP DBD line 2. As a control, an extract from a nontransgenic thymus was loaded. The PARP DBD protein was detected with the monoclonal antibody CII10, which recognizes the hPARP transgene and the endogenous mPARP. The relative amount of PARP DBD protein expression in splenocytes is detectable but very low (data not shown).

    Techniques Used: Dominant Negative Mutation, Transgenic Assay, Mouse Assay, Construct, Expressing, Software, Immunofluorescence

    Thymocytes expressing PARP DBD or lacking the PARP gene are more sensitive to apoptosis upon DNA damage only in the presence of p53. (a to c) Thymocytes were explanted from lck -PARP DBD transgenic mice and from nontransgenic (non tg) controls and were either irradiated (2 Gy) (a) or treated with MNNG (40 μM) (b) or etoposide (50 μM) (c). Before treatment and at different time points thereafter, samples were analyzed for the percentage of apoptotic cells. Shown are mean values with standard deviations representative of three independent sets of experiments. Each data point represents an average value with standard deviation obtained from measurements of three different mice. (d) Thymocytes were explanted from PARP null mice (PARP −/− ), from mutant mice that are PARP null and express the PARP DBD (PARP −/− , lck-PARP-DBD) from mice that are deficient for both PARP and p53 (PARP −/− , p53 −/− ), and from nontransgenic controls and were irradiated with a single pulse of 2 Gy. Before treatment and at different times thereafter, samples were analyzed for the percentage of apoptotic cells. Shown are mean values with standard deviations representative of three independent sets of experiments. Each data point represents an average value with standard deviation obtained from measurements of three different mice.
    Figure Legend Snippet: Thymocytes expressing PARP DBD or lacking the PARP gene are more sensitive to apoptosis upon DNA damage only in the presence of p53. (a to c) Thymocytes were explanted from lck -PARP DBD transgenic mice and from nontransgenic (non tg) controls and were either irradiated (2 Gy) (a) or treated with MNNG (40 μM) (b) or etoposide (50 μM) (c). Before treatment and at different time points thereafter, samples were analyzed for the percentage of apoptotic cells. Shown are mean values with standard deviations representative of three independent sets of experiments. Each data point represents an average value with standard deviation obtained from measurements of three different mice. (d) Thymocytes were explanted from PARP null mice (PARP −/− ), from mutant mice that are PARP null and express the PARP DBD (PARP −/− , lck-PARP-DBD) from mice that are deficient for both PARP and p53 (PARP −/− , p53 −/− ), and from nontransgenic controls and were irradiated with a single pulse of 2 Gy. Before treatment and at different times thereafter, samples were analyzed for the percentage of apoptotic cells. Shown are mean values with standard deviations representative of three independent sets of experiments. Each data point represents an average value with standard deviation obtained from measurements of three different mice.

    Techniques Used: Expressing, Transgenic Assay, Mouse Assay, Irradiation, Standard Deviation, Mutagenesis

    Accelerated apoptosis in thymocytes from lck -PARP DBD transgenic mice is reflected by earlier cleavage of endogenous PARP and increased DNA laddering. (a) To monitor endogenous PARP cleavage, thymocytes of lck -PARP DBD transgenic mice and nontransgenic (non tg) littermates were cultured and either left untreated or irradiated with a single dose of 2 Gy. Samples were taken at the indicated time points after the stimulus and were analyzed by Western blotting. Equal loading of each lane was controlled by Ponceau staining (not shown). (b) To confirm that both X irradiation and treatment with MNNG induce apoptosis, genomic DNA prepared from thymocytes of lck -PARP DBD transgenic and nontransgenic control mice after treatment with MNNG was analyzed on agarose gels. The presence of nucleosomal DNA degradation indicates apoptosis.
    Figure Legend Snippet: Accelerated apoptosis in thymocytes from lck -PARP DBD transgenic mice is reflected by earlier cleavage of endogenous PARP and increased DNA laddering. (a) To monitor endogenous PARP cleavage, thymocytes of lck -PARP DBD transgenic mice and nontransgenic (non tg) littermates were cultured and either left untreated or irradiated with a single dose of 2 Gy. Samples were taken at the indicated time points after the stimulus and were analyzed by Western blotting. Equal loading of each lane was controlled by Ponceau staining (not shown). (b) To confirm that both X irradiation and treatment with MNNG induce apoptosis, genomic DNA prepared from thymocytes of lck -PARP DBD transgenic and nontransgenic control mice after treatment with MNNG was analyzed on agarose gels. The presence of nucleosomal DNA degradation indicates apoptosis.

    Techniques Used: Transgenic Assay, Mouse Assay, DNA Laddering, Cell Culture, Irradiation, Western Blot, Staining

    Inhibition of poly(ADP-ribosyl)ation inhibits DNA strand break resealing upon MNNG treatment or X irradiation. Comet assays were performed to assess the effects of PARP DBD expression on the resealing of DNA strand breaks. Thymocytes of lck -PARP DBD transgenic mice and age-matched nontransgenic (non tg) control littermates were prepared, treated with 40 μM MNNG for 20 min, stained with propidium iodide, and analyzed for comet formation. Given are average values with standard deviations from 40 cells per sample and time point against elapsed time after the stimulus. In each case, a representative of three independent experiments is depicted. For measurement of the comets, a fluorescence microscope was coupled with an intensified target camera, and an interactive digital image analysis system was established.
    Figure Legend Snippet: Inhibition of poly(ADP-ribosyl)ation inhibits DNA strand break resealing upon MNNG treatment or X irradiation. Comet assays were performed to assess the effects of PARP DBD expression on the resealing of DNA strand breaks. Thymocytes of lck -PARP DBD transgenic mice and age-matched nontransgenic (non tg) control littermates were prepared, treated with 40 μM MNNG for 20 min, stained with propidium iodide, and analyzed for comet formation. Given are average values with standard deviations from 40 cells per sample and time point against elapsed time after the stimulus. In each case, a representative of three independent experiments is depicted. For measurement of the comets, a fluorescence microscope was coupled with an intensified target camera, and an interactive digital image analysis system was established.

    Techniques Used: Inhibition, Irradiation, Expressing, Transgenic Assay, Mouse Assay, Staining, Fluorescence, Microscopy

    Expression and activity of p53 upon DNA damage depend on poly(ADP-ribosyl)ation. (a) Luciferase activity (in relative light units [RLU]) after transient transfection of EL-4 T cells with the lck -driven PARP DBD (lck-PARP-DBD) or the empty lck vector. Transcriptional transactivation of the MDM2 reporter was measured in untreated cells or upon treatment with MNNG (left) or after X irradiation (2 Gy; right). Given are average values with standard deviations from three independent transfections. (b) Immunoblot analysis of p53 expression levels. EL-4 T cells were transiently transfected with either the empty lck vector or the lck -PARP DBD expression construct and left untreated or irradiated (2 Gy). After 4 h of exposure to the stimulus, cell extracts were prepared and analyzed by Western blotting. Equal loading of each lane was controlled by Ponceau staining (not shown).
    Figure Legend Snippet: Expression and activity of p53 upon DNA damage depend on poly(ADP-ribosyl)ation. (a) Luciferase activity (in relative light units [RLU]) after transient transfection of EL-4 T cells with the lck -driven PARP DBD (lck-PARP-DBD) or the empty lck vector. Transcriptional transactivation of the MDM2 reporter was measured in untreated cells or upon treatment with MNNG (left) or after X irradiation (2 Gy; right). Given are average values with standard deviations from three independent transfections. (b) Immunoblot analysis of p53 expression levels. EL-4 T cells were transiently transfected with either the empty lck vector or the lck -PARP DBD expression construct and left untreated or irradiated (2 Gy). After 4 h of exposure to the stimulus, cell extracts were prepared and analyzed by Western blotting. Equal loading of each lane was controlled by Ponceau staining (not shown).

    Techniques Used: Expressing, Activity Assay, Luciferase, Transfection, Plasmid Preparation, Irradiation, Construct, Western Blot, Staining

    Influence of p53 and PARP on DNA resealing after induction of damage by MNNG treatment and X irradiation. (a) Comet assay to compare DNA repair kinetics after MNNG treatment between cells from lck -PARP DBD mice, p53-deficient animals, mice that are both p53 deficient and carry an lck -PARP DBD transgene, and age-matched nontransgenic (non tg) littermate controls. (b) Comet assay to compare DNA repair kinetics after X irradiation with a single dose of 2 Gy between cells from lck -PARP DBD mice, p53-deficient animals, mice that are both p53 deficient and carry an lck -PARP DBD transgene, and age-matched nontransgenic littermate controls. Given are average values with standard deviations from 40 cells per sample and time point against elapsed time after the stimulus. In each case, a representative of three independent experiments is depicted. For measurement of the comets, a fluorescence microscope was coupled with an intensified target camera, and an interactive digital image analysis system was established.
    Figure Legend Snippet: Influence of p53 and PARP on DNA resealing after induction of damage by MNNG treatment and X irradiation. (a) Comet assay to compare DNA repair kinetics after MNNG treatment between cells from lck -PARP DBD mice, p53-deficient animals, mice that are both p53 deficient and carry an lck -PARP DBD transgene, and age-matched nontransgenic (non tg) littermate controls. (b) Comet assay to compare DNA repair kinetics after X irradiation with a single dose of 2 Gy between cells from lck -PARP DBD mice, p53-deficient animals, mice that are both p53 deficient and carry an lck -PARP DBD transgene, and age-matched nontransgenic littermate controls. Given are average values with standard deviations from 40 cells per sample and time point against elapsed time after the stimulus. In each case, a representative of three independent experiments is depicted. For measurement of the comets, a fluorescence microscope was coupled with an intensified target camera, and an interactive digital image analysis system was established.

    Techniques Used: Irradiation, Single Cell Gel Electrophoresis, Mouse Assay, Fluorescence, Microscopy

    9) Product Images from "α-Lipoic acid prevents the intestinal epithelial monolayer damage under heat stress conditions: model experiments in Caco-2 cells"

    Article Title: α-Lipoic acid prevents the intestinal epithelial monolayer damage under heat stress conditions: model experiments in Caco-2 cells

    Journal: European Journal of Nutrition

    doi: 10.1007/s00394-017-1442-y

    ALA partly prevents the HS-induced disturbance of E-cadherin expression. Caco-2 cells grown on inserts and pretreated with ALA (24 h) were exposed to HS (42 °C) for 24 h to evaluate the E-cadherin protein expression ( a ) and cellular distribution ( b ). For the WB analysis, results are expressed as protein expression (normalized with β-actin) relative to unstimulated cells as mean ± SEM of three independent experiments. Different lower case letters denote significant differences among groups. Localization of E-cadherin was evaluated by immunofluorescence staining (Objective, ×40)
    Figure Legend Snippet: ALA partly prevents the HS-induced disturbance of E-cadherin expression. Caco-2 cells grown on inserts and pretreated with ALA (24 h) were exposed to HS (42 °C) for 24 h to evaluate the E-cadherin protein expression ( a ) and cellular distribution ( b ). For the WB analysis, results are expressed as protein expression (normalized with β-actin) relative to unstimulated cells as mean ± SEM of three independent experiments. Different lower case letters denote significant differences among groups. Localization of E-cadherin was evaluated by immunofluorescence staining (Objective, ×40)

    Techniques Used: Expressing, Western Blot, Immunofluorescence, Staining

    ALA stimulates the restitution of epithelial wound healing. Confluent Caco-2 cells grown in 6-well plates and pretreated with ALA (24 h) were scratched with a 200-µl pipette tip and were exposed to control or HS (42 °C) conditions for 24 h. Phase contrast images were acquired immediately after scratching (0 h) and 24 h thereafter. Wound widths are expressed as percentage of initial value as mean ± SEM of three independent experiments. Different lower case letters denote significant differences among groups
    Figure Legend Snippet: ALA stimulates the restitution of epithelial wound healing. Confluent Caco-2 cells grown in 6-well plates and pretreated with ALA (24 h) were scratched with a 200-µl pipette tip and were exposed to control or HS (42 °C) conditions for 24 h. Phase contrast images were acquired immediately after scratching (0 h) and 24 h thereafter. Wound widths are expressed as percentage of initial value as mean ± SEM of three independent experiments. Different lower case letters denote significant differences among groups

    Techniques Used: Transferring

    ALA prevents the HS-induced upregulation of TGF-β and COX-2 mRNA expression. Caco-2 cells grown on inserts and pretreated with ALA (24 h) were exposed to HS (42 °C, 6h) to evaluate the mRNA expression of TGF-β and COX-2 (qRT-PCR). Results are expressed as mRNA expression (normalized with β-actin) relative to unstimulated cells as mean ± SEM of three independent experiments. Different lower case letters denote significant differences among groups
    Figure Legend Snippet: ALA prevents the HS-induced upregulation of TGF-β and COX-2 mRNA expression. Caco-2 cells grown on inserts and pretreated with ALA (24 h) were exposed to HS (42 °C, 6h) to evaluate the mRNA expression of TGF-β and COX-2 (qRT-PCR). Results are expressed as mRNA expression (normalized with β-actin) relative to unstimulated cells as mean ± SEM of three independent experiments. Different lower case letters denote significant differences among groups

    Techniques Used: Expressing, Quantitative RT-PCR

    HS upregulates the HSF1 protein expression and induces HSF1 nuclear granules. Caco-2 cells grown on inserts and pretreated with ALA (24 h) were exposed to HS (42 °C, 6 h). HSF1 protein expression (normalized with β-actin) relative to unstimulated cells evaluated by WB analysis ( a ), is expressed as mean ± SEM of three independent experiments. Different lower case letters denote significant differences among groups. Localization of HSF1 was visualized by immunofluorescence staining. Objective ×40 ( b ) and ×63 ( c )
    Figure Legend Snippet: HS upregulates the HSF1 protein expression and induces HSF1 nuclear granules. Caco-2 cells grown on inserts and pretreated with ALA (24 h) were exposed to HS (42 °C, 6 h). HSF1 protein expression (normalized with β-actin) relative to unstimulated cells evaluated by WB analysis ( a ), is expressed as mean ± SEM of three independent experiments. Different lower case letters denote significant differences among groups. Localization of HSF1 was visualized by immunofluorescence staining. Objective ×40 ( b ) and ×63 ( c )

    Techniques Used: Expressing, Western Blot, Immunofluorescence, Staining

    ALA increases the HSP70 expression under HS conditions. Caco-2 cells grown on inserts and pretreated with ALA (24h) were exposed to HS (42 °C) for 6 h (qRT-PCR) or 24 h (WB) to evaluate the expression of HSP70 in mRNA ( a ) and protein levels ( b ). Results are expressed as mRNA expression or protein expression (normalized with β-actin) relative to unstimulated cells as mean ± SEM of three independent experiments. Different lower case letters denote significant differences among groups
    Figure Legend Snippet: ALA increases the HSP70 expression under HS conditions. Caco-2 cells grown on inserts and pretreated with ALA (24h) were exposed to HS (42 °C) for 6 h (qRT-PCR) or 24 h (WB) to evaluate the expression of HSP70 in mRNA ( a ) and protein levels ( b ). Results are expressed as mRNA expression or protein expression (normalized with β-actin) relative to unstimulated cells as mean ± SEM of three independent experiments. Different lower case letters denote significant differences among groups

    Techniques Used: Expressing, Quantitative RT-PCR, Western Blot

    ALA prevents the HS-induced disruption of epithelial integrity and accelerates the JC reassembly. Caco-2 cells grown on inserts were pretreated with ALA (24 h) prior to HS exposure (42 °C) and TEER ( a ) and LY transport ( b ) across the Caco-2 monolayer was measured after 24 h exposure to HS. For the calcium switch assay, Caco-2 cells were pretreated with ALA (24 h) before calcium deprivation and TEER was measured during recovery (0, 2, 4, 6, 8 h) in medium supplemented with ALA ( c ). Data are expressed as a percentage of initial value (TEER, calcium switch assay) or in the amount LY transported [ng/(cm 2 × h)] detected in the basolateral compartment of transwell inserts as mean ± SEM of three independent experiments. Different lower case letters denote significant differences among groups
    Figure Legend Snippet: ALA prevents the HS-induced disruption of epithelial integrity and accelerates the JC reassembly. Caco-2 cells grown on inserts were pretreated with ALA (24 h) prior to HS exposure (42 °C) and TEER ( a ) and LY transport ( b ) across the Caco-2 monolayer was measured after 24 h exposure to HS. For the calcium switch assay, Caco-2 cells were pretreated with ALA (24 h) before calcium deprivation and TEER was measured during recovery (0, 2, 4, 6, 8 h) in medium supplemented with ALA ( c ). Data are expressed as a percentage of initial value (TEER, calcium switch assay) or in the amount LY transported [ng/(cm 2 × h)] detected in the basolateral compartment of transwell inserts as mean ± SEM of three independent experiments. Different lower case letters denote significant differences among groups

    Techniques Used:

    ALA prevents the HS-induced expression and nuclear translocation of Nrf2. Caco-2 cells grown on inserts (qRT-PCR) or 6-well plates (WB) and pretreated with ALA (24 h) were exposed to HS (42 °C) for 6 h. Results are expressed as mRNA expression (normalized with β-actin) ( a ) and nuclear abundance (normalized with Lamin A) ( b ) relative to unstimulated cells as mean ± SEM of three independent experiments. Different lower case letters denote significant differences among groups
    Figure Legend Snippet: ALA prevents the HS-induced expression and nuclear translocation of Nrf2. Caco-2 cells grown on inserts (qRT-PCR) or 6-well plates (WB) and pretreated with ALA (24 h) were exposed to HS (42 °C) for 6 h. Results are expressed as mRNA expression (normalized with β-actin) ( a ) and nuclear abundance (normalized with Lamin A) ( b ) relative to unstimulated cells as mean ± SEM of three independent experiments. Different lower case letters denote significant differences among groups

    Techniques Used: Expressing, Translocation Assay, Quantitative RT-PCR, Western Blot

    10) Product Images from "Citrullination of Histone H3 Interferes with HP1-Mediated Transcriptional Repression"

    Article Title: Citrullination of Histone H3 Interferes with HP1-Mediated Transcriptional Repression

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1002934

    PADI4 activity controls HP1α occupancy and histone H3 citrullination at TNFα and HERV promoters. (A) Total protein extracts from MCF7 cells treated either estradiol (E2) or ionophore (A23187) and/or Cl-amidine were immunoblotted with the indicated antibodies. (B) Total RNA from MCF7 cells either untreated or treated with estradiol followed by ionophore (E2+A23187) and/or Cl-amidine (E2+A23187+Cl-amidine) treatment was quantified by RT-qPCR. Data are shown relative to the un-induced condition (set to 1). Values are mean ± SEM from four experimental replicates. (C–E) ChIP with the listed antibodies was carried out with chromatin from MCF7 cells either untreated or treated with estradiol and ionophore (E2+A23187) in the absence or presence of Cl-amidine. The relative enrichments of the indicated antibodies on the shown LTRs or promoters were measured by qPCR. Data are presented as a percentage of histone H3 or relative to non-immune IgG as indicated. Enrichments are presented relative to indicated controls (set to 1). Values are means ± SEM from four PCR measures of representative ChIP experiments.
    Figure Legend Snippet: PADI4 activity controls HP1α occupancy and histone H3 citrullination at TNFα and HERV promoters. (A) Total protein extracts from MCF7 cells treated either estradiol (E2) or ionophore (A23187) and/or Cl-amidine were immunoblotted with the indicated antibodies. (B) Total RNA from MCF7 cells either untreated or treated with estradiol followed by ionophore (E2+A23187) and/or Cl-amidine (E2+A23187+Cl-amidine) treatment was quantified by RT-qPCR. Data are shown relative to the un-induced condition (set to 1). Values are mean ± SEM from four experimental replicates. (C–E) ChIP with the listed antibodies was carried out with chromatin from MCF7 cells either untreated or treated with estradiol and ionophore (E2+A23187) in the absence or presence of Cl-amidine. The relative enrichments of the indicated antibodies on the shown LTRs or promoters were measured by qPCR. Data are presented as a percentage of histone H3 or relative to non-immune IgG as indicated. Enrichments are presented relative to indicated controls (set to 1). Values are means ± SEM from four PCR measures of representative ChIP experiments.

    Techniques Used: Activity Assay, Quantitative RT-PCR, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction

    11) Product Images from "Intra- and Intermolecular Disulfide Bonds of the GP2b Glycoprotein of Equine Arteritis Virus: Relevance for Virus Assembly and Infectivity"

    Article Title: Intra- and Intermolecular Disulfide Bonds of the GP2b Glycoprotein of Equine Arteritis Virus: Relevance for Virus Assembly and Infectivity

    Journal: Journal of Virology

    doi: 10.1128/JVI.77.24.12996-13004.2003

    Conformational variants of the GP 2b monomers. vTF7.3-infected BHK-21 C13 cells were transfected with plasmids encoding the wild-type GP 2b protein or the GP 2b cysteine mutants. At 4 h p.i., the cells were labeled for 15 min with [ 35 S]methionine. Next, cell lysates were prepared in the presence of IAM and immunoprecipitations were performed with αGP 2b . The immunoprecipitates were analyzed under nonreducing conditions in an SDS-15% PAA gel. The arrowheads indicate the positions of the different GP 2b conformations. The values on the left are the molecular sizes, in kilodaltons, of marker proteins analyzed in the same gel.
    Figure Legend Snippet: Conformational variants of the GP 2b monomers. vTF7.3-infected BHK-21 C13 cells were transfected with plasmids encoding the wild-type GP 2b protein or the GP 2b cysteine mutants. At 4 h p.i., the cells were labeled for 15 min with [ 35 S]methionine. Next, cell lysates were prepared in the presence of IAM and immunoprecipitations were performed with αGP 2b . The immunoprecipitates were analyzed under nonreducing conditions in an SDS-15% PAA gel. The arrowheads indicate the positions of the different GP 2b conformations. The values on the left are the molecular sizes, in kilodaltons, of marker proteins analyzed in the same gel.

    Techniques Used: Infection, Transfection, Labeling, Marker

    12) Product Images from "TRE17/USP6 oncogene translocated in aneurysmal bone cyst induces matrix metalloproteinase production via activation of NF?B"

    Article Title: TRE17/USP6 oncogene translocated in aneurysmal bone cyst induces matrix metalloproteinase production via activation of NF?B

    Journal: Oncogene

    doi: 10.1038/onc.2010.116

    RhoA and ROCK partially mediate activation of NFκB by TRE17 (A) RhoA activity was measured in transiently transfected hFOB1.19 (left), HeLa (middle), and MC3T3 cell lines (right). Cell extracts were subjected to pulldowns using a GST fusion of the RhoA·GTP-binding domain (RBD) of Rhotekin, followed by blotting with anti-RhoA. Active RhoA, RBD pulldowns; total RhoA, whole cell lysates. (B) hFOB1.19 or HeLa were transfected with NFκB-luciferase and TRE17(long), in the absence or presence of dominant negative RhoA (RhoAN19) or Y27632, and subjected to luciferase assays. (C) Control or TRE17(long)/MC3T3 cells were treated with dox for 24 hrs, then starved with dox and the indicated concentration of Y27632 (μM) for 24 hrs. MMP-9 in the CM was detected by immunoblotting and zymography. Whole cell extracts were blotted to confirm TRE17 and actin levels.
    Figure Legend Snippet: RhoA and ROCK partially mediate activation of NFκB by TRE17 (A) RhoA activity was measured in transiently transfected hFOB1.19 (left), HeLa (middle), and MC3T3 cell lines (right). Cell extracts were subjected to pulldowns using a GST fusion of the RhoA·GTP-binding domain (RBD) of Rhotekin, followed by blotting with anti-RhoA. Active RhoA, RBD pulldowns; total RhoA, whole cell lysates. (B) hFOB1.19 or HeLa were transfected with NFκB-luciferase and TRE17(long), in the absence or presence of dominant negative RhoA (RhoAN19) or Y27632, and subjected to luciferase assays. (C) Control or TRE17(long)/MC3T3 cells were treated with dox for 24 hrs, then starved with dox and the indicated concentration of Y27632 (μM) for 24 hrs. MMP-9 in the CM was detected by immunoblotting and zymography. Whole cell extracts were blotted to confirm TRE17 and actin levels.

    Techniques Used: Activation Assay, Activity Assay, Transfection, Binding Assay, Luciferase, Dominant Negative Mutation, Concentration Assay, Zymography

    TRE17 induces MMP expression on a transcriptional level (A) hFOB1.19 or (B) HeLa cells were transfected, then serum-starved for 24-48 hours. MC3T3-derived cell lines (C) were treated with or without dox for 24 hrs, then starved for an additional 24 hrs. For all three cell systems RNA was isolated, and RT-qPCR was performed to monitor MMP-9 and MMP-10 mRNA, using two independent primer pairs for each (denoted MMP9-1 and -2, and MMP10-1 and -2), normalizing against GAPDH. For hFOB1.19 and HeLa cells, data represent the mean ± SD of three experiments. Samples with statistically significant differences from vector control cells are indicated (*, p
    Figure Legend Snippet: TRE17 induces MMP expression on a transcriptional level (A) hFOB1.19 or (B) HeLa cells were transfected, then serum-starved for 24-48 hours. MC3T3-derived cell lines (C) were treated with or without dox for 24 hrs, then starved for an additional 24 hrs. For all three cell systems RNA was isolated, and RT-qPCR was performed to monitor MMP-9 and MMP-10 mRNA, using two independent primer pairs for each (denoted MMP9-1 and -2, and MMP10-1 and -2), normalizing against GAPDH. For hFOB1.19 and HeLa cells, data represent the mean ± SD of three experiments. Samples with statistically significant differences from vector control cells are indicated (*, p

    Techniques Used: Expressing, Transfection, Derivative Assay, Isolation, Quantitative RT-PCR, Plasmid Preparation

    NFκB is required for induction of MMP-9 by TRE17 A) hFOB1.19 or HeLa cells were transfected with the indicated TRE17 constructs and either a luciferase reporter driven by the WT MMP-9 promoter, or a mutant in which the NFκB response element was ablated. Luciferase assays were performed; data represent the mean ± SD of 4 experiments (*, p
    Figure Legend Snippet: NFκB is required for induction of MMP-9 by TRE17 A) hFOB1.19 or HeLa cells were transfected with the indicated TRE17 constructs and either a luciferase reporter driven by the WT MMP-9 promoter, or a mutant in which the NFκB response element was ablated. Luciferase assays were performed; data represent the mean ± SD of 4 experiments (*, p

    Techniques Used: Transfection, Construct, Luciferase, Mutagenesis

    TRE17(long) induces formation of tumors that recapitulate multiple features of ABC in a USP-dependent manner (A) In vivo bioluminescence imaging of mice injected with MC3T3 cell lines expressing TRE17(long), TRE17(long)/USP-, or luciferase alone. ( B ) Left, macroscopic images of tumor and injection site of mice injected with vector or TRE17(long)-expressing MC3T3. Right, H E stain of TRE17(long) tumor section. Arrowheads highlight vascular elements. ( C ) Immunohistochemistry was performed on serial sections of TRE17(long)-induced tumor using antibodies against TRE17 and MMP-9. Nuclear staining for TRE17 is non-specific; only central cells exhibit specific cytoplasmic staining of TRE17.
    Figure Legend Snippet: TRE17(long) induces formation of tumors that recapitulate multiple features of ABC in a USP-dependent manner (A) In vivo bioluminescence imaging of mice injected with MC3T3 cell lines expressing TRE17(long), TRE17(long)/USP-, or luciferase alone. ( B ) Left, macroscopic images of tumor and injection site of mice injected with vector or TRE17(long)-expressing MC3T3. Right, H E stain of TRE17(long) tumor section. Arrowheads highlight vascular elements. ( C ) Immunohistochemistry was performed on serial sections of TRE17(long)-induced tumor using antibodies against TRE17 and MMP-9. Nuclear staining for TRE17 is non-specific; only central cells exhibit specific cytoplasmic staining of TRE17.

    Techniques Used: In Vivo, Imaging, Mouse Assay, Injection, Expressing, Luciferase, Plasmid Preparation, Staining, Immunohistochemistry

    TRE17 induces secretion of MMP-9 and MMP-10 in a USP-dependent manner (A) hFOB1.19 human fetal osteoblasts were transfected with the indicated HA-tagged TRE17 constructs, then serum-starved. MMP-9 and MMP-10 in the CM were concentrated as described in Experimental Procedures, and detected by immunoblotting. Whole cell lysates were blotted for TRE17 (arrowheads indicate migration of TRE17(long) and TRE17(short) isoforms; asterisk denotes non-specific (ns) band recognized by antibody), and actin as a loading control. ( B) Domain structure of TRE17 alleles. TBC, TBC domain; C and H, cysteine and histidine subdomains of the USP domain. TRE17(long)/USP- and TRE17/A6- harbor point mutations in the indicated domains, as detailed in Experimental Procedures. (C) MC3T3 osteoblasts stably expressing HA-tagged TRE17(long) or TRE17(A6-) in a doxycycline (dox)-inducible manner were treated with dox (2 μg/ml) for 24 hrs. Cell extracts were immunoprecipitated with anti-HA, then blotted with anti-Arf6 (top panel) or anti-TRE17 (bottom panel). I.P., anti-HA immunoprecipitate; WCL, whole cell lysate. (D) Stable MC3T3 cell lines expressing the indicated TRE17 alleles were grown with or without dox for 24 hrs, then starved for 24 hrs in the continued absence or presence of dox. MMP-9 was purified from the CM then subjected to blotting, or zymography to detect gelatinolytic activity. (E) HeLa cells were transfected as indicated, and accumulation of MMP-9 and MMP-10 in the CM was monitored as in (A).
    Figure Legend Snippet: TRE17 induces secretion of MMP-9 and MMP-10 in a USP-dependent manner (A) hFOB1.19 human fetal osteoblasts were transfected with the indicated HA-tagged TRE17 constructs, then serum-starved. MMP-9 and MMP-10 in the CM were concentrated as described in Experimental Procedures, and detected by immunoblotting. Whole cell lysates were blotted for TRE17 (arrowheads indicate migration of TRE17(long) and TRE17(short) isoforms; asterisk denotes non-specific (ns) band recognized by antibody), and actin as a loading control. ( B) Domain structure of TRE17 alleles. TBC, TBC domain; C and H, cysteine and histidine subdomains of the USP domain. TRE17(long)/USP- and TRE17/A6- harbor point mutations in the indicated domains, as detailed in Experimental Procedures. (C) MC3T3 osteoblasts stably expressing HA-tagged TRE17(long) or TRE17(A6-) in a doxycycline (dox)-inducible manner were treated with dox (2 μg/ml) for 24 hrs. Cell extracts were immunoprecipitated with anti-HA, then blotted with anti-Arf6 (top panel) or anti-TRE17 (bottom panel). I.P., anti-HA immunoprecipitate; WCL, whole cell lysate. (D) Stable MC3T3 cell lines expressing the indicated TRE17 alleles were grown with or without dox for 24 hrs, then starved for 24 hrs in the continued absence or presence of dox. MMP-9 was purified from the CM then subjected to blotting, or zymography to detect gelatinolytic activity. (E) HeLa cells were transfected as indicated, and accumulation of MMP-9 and MMP-10 in the CM was monitored as in (A).

    Techniques Used: Transfection, Construct, Migration, Stable Transfection, Expressing, Immunoprecipitation, Purification, Zymography, Activity Assay

    13) Product Images from "cjrABC-senB hinders survival of extraintestinal pathogenic E. coli in the bloodstream through triggering complement-mediated killing"

    Article Title: cjrABC-senB hinders survival of extraintestinal pathogenic E. coli in the bloodstream through triggering complement-mediated killing

    Journal: Journal of Biomedical Science

    doi: 10.1186/s12929-020-00677-4

    The fold difference in the serum survival of Cjr − -RS218 compared to that of Cjr + -RS218 and the deposition of C1q and properdin on the strains. ( a ) The fold difference in the serum survival (the survival rate of Cjr − -RS218/the survival rate of Cjr + -RS218) after 3 h of incubation in 40% sera, which were NHS and NHS with the classical, alternative, or lectin pathway inhibited. The horizontal dashed line represents 1-fold (the survival of the two strains is similar). The serum survival of Cjr − -RS218 was approximately 24.8-, 1.1-, 1.5-, and 22.9-fold greater than that of Cjr + -RS218 in NHS or NHS in which the classical, alternative, or lectin pathway was inhibited, respectively. ( b ) The fold difference in survival in 40% HI-NHS and 40%-modified HI-NHS, in which the classical, alternative, or lectin pathway was inhibited. Cjr − -RS218 and Cjr + -RS218 showed similar serum survival in these sera. For ( a ) and ( b ), the results are shown as the mean ± standard deviation, and the data are representative of three independent experiments performed in triplicate. Classical − NHS, C1q-depleted NHS in which the CP is blocked; Alternative − NHS, factor B-depleted NHS in which the AP is blocked; Lectin − NHS, mannose-treated NHS in which the LP is blocked; Classical − HI-NHS, heat-inactivated C1q-depleted NHS; Alternative − HI-NHS, heat-inactivated factor B-depleted NHS; Lectin − HI-NHS, heat-inactivated mannose-treated NHS. ( c ) The levels of C1q deposition after incubation in 40% NHS. ( d ) Flow cytometry histogram of C1q deposition on the bacteria after 3 h of incubation in NHS. ( e ) The levels of properdin deposition after incubation in 40% NHS. ( f ) Flow cytometry histogram of properdin deposition on the bacteria after 3 h of incubation in NHS. For ( c ) and ( e ), the data are presented with GMFI. The results are shown as the means ± standard deviations, and the data are derived from three independent experiments. The HI-NHS control groups were bacteria incubated in HI-NHS, while the unstaining control groups were the bacteria without fluorescence staining. *, P value
    Figure Legend Snippet: The fold difference in the serum survival of Cjr − -RS218 compared to that of Cjr + -RS218 and the deposition of C1q and properdin on the strains. ( a ) The fold difference in the serum survival (the survival rate of Cjr − -RS218/the survival rate of Cjr + -RS218) after 3 h of incubation in 40% sera, which were NHS and NHS with the classical, alternative, or lectin pathway inhibited. The horizontal dashed line represents 1-fold (the survival of the two strains is similar). The serum survival of Cjr − -RS218 was approximately 24.8-, 1.1-, 1.5-, and 22.9-fold greater than that of Cjr + -RS218 in NHS or NHS in which the classical, alternative, or lectin pathway was inhibited, respectively. ( b ) The fold difference in survival in 40% HI-NHS and 40%-modified HI-NHS, in which the classical, alternative, or lectin pathway was inhibited. Cjr − -RS218 and Cjr + -RS218 showed similar serum survival in these sera. For ( a ) and ( b ), the results are shown as the mean ± standard deviation, and the data are representative of three independent experiments performed in triplicate. Classical − NHS, C1q-depleted NHS in which the CP is blocked; Alternative − NHS, factor B-depleted NHS in which the AP is blocked; Lectin − NHS, mannose-treated NHS in which the LP is blocked; Classical − HI-NHS, heat-inactivated C1q-depleted NHS; Alternative − HI-NHS, heat-inactivated factor B-depleted NHS; Lectin − HI-NHS, heat-inactivated mannose-treated NHS. ( c ) The levels of C1q deposition after incubation in 40% NHS. ( d ) Flow cytometry histogram of C1q deposition on the bacteria after 3 h of incubation in NHS. ( e ) The levels of properdin deposition after incubation in 40% NHS. ( f ) Flow cytometry histogram of properdin deposition on the bacteria after 3 h of incubation in NHS. For ( c ) and ( e ), the data are presented with GMFI. The results are shown as the means ± standard deviations, and the data are derived from three independent experiments. The HI-NHS control groups were bacteria incubated in HI-NHS, while the unstaining control groups were the bacteria without fluorescence staining. *, P value

    Techniques Used: Incubation, Modification, Standard Deviation, Flow Cytometry, Derivative Assay, Fluorescence, Staining

    14) Product Images from "The Envelope Cytoplasmic Tail of HIV-1 Subtype C Contributes to Poor Replication Capacity through Low Viral Infectivity and Cell-to-Cell Transmission"

    Article Title: The Envelope Cytoplasmic Tail of HIV-1 Subtype C Contributes to Poor Replication Capacity through Low Viral Infectivity and Cell-to-Cell Transmission

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0161596

    The gp41CT of subtype C Envs does not tether virus to CD4+ T-cells. CD4+ T-cells (10 5 cells/well) were infected with Env or EnvEC recombinant virus pairs for 5 days, treated with subtilisin or with buffer alone, and virus in each fraction was measured by p24 ELISA. The amount of p24 that released upon subtilisin-treatment was reported to total p24 (i.e. released in the supernatant+virus attached to the PM). Three experiments with CD4+ T-cells from different donors are shown (average). Error bars represent standard deviation.
    Figure Legend Snippet: The gp41CT of subtype C Envs does not tether virus to CD4+ T-cells. CD4+ T-cells (10 5 cells/well) were infected with Env or EnvEC recombinant virus pairs for 5 days, treated with subtilisin or with buffer alone, and virus in each fraction was measured by p24 ELISA. The amount of p24 that released upon subtilisin-treatment was reported to total p24 (i.e. released in the supernatant+virus attached to the PM). Three experiments with CD4+ T-cells from different donors are shown (average). Error bars represent standard deviation.

    Techniques Used: Infection, Recombinant, Enzyme-linked Immunosorbent Assay, Standard Deviation

    15) Product Images from "CLAG3 Self-Associates in Malaria Parasites and Quantitatively Determines Nutrient Uptake Channels at the Host Membrane"

    Article Title: CLAG3 Self-Associates in Malaria Parasites and Quantitatively Determines Nutrient Uptake Channels at the Host Membrane

    Journal: mBio

    doi: 10.1128/mBio.02293-17

    Coexpression and faithful trafficking of tandem CLAG3 isoforms. (A) Indirect immunofluorescence images showing colocalization of Dd2-HA and 3D7-miniSOG-FLAG CLAG3 isoforms in TFLC3 parasites at the schizont stage and in trophozoites after invasion of new erythrocytes. Bar, 5 µm. (B and C) Immunoblot assays performed using TFLC3 cell lysates and indicated epitope tag antibodies without or with extracellular pronase E treatment prior to cell lysis (− and +, respectively). (D and E) Immunoblot assays using anti-CLAG3 antibody and indicated parasites, without and with pronase E treatment (− and +, respectively). While two cleavage products are detected in TFLC3 , the C3attB parent produces a single fragment.
    Figure Legend Snippet: Coexpression and faithful trafficking of tandem CLAG3 isoforms. (A) Indirect immunofluorescence images showing colocalization of Dd2-HA and 3D7-miniSOG-FLAG CLAG3 isoforms in TFLC3 parasites at the schizont stage and in trophozoites after invasion of new erythrocytes. Bar, 5 µm. (B and C) Immunoblot assays performed using TFLC3 cell lysates and indicated epitope tag antibodies without or with extracellular pronase E treatment prior to cell lysis (− and +, respectively). (D and E) Immunoblot assays using anti-CLAG3 antibody and indicated parasites, without and with pronase E treatment (− and +, respectively). While two cleavage products are detected in TFLC3 , the C3attB parent produces a single fragment.

    Techniques Used: Immunofluorescence, Lysis

    CLAG3 merodiploids reveal a stoichiometric contribution to PSAC-mediated nutrient uptake. (A) Schematic showing the minimum distinct combinations of host membrane RhopH complexes in the TFLC3 parasite. CLAG3 is shown as blue ribbon dimers embedded in the host membrane; note the permutations of the two C-terminal epitope tags (gold circles and pink ellipses). RhopH2 and RhopH3 are represented in green. (B) Sorbitol uptake kinetics for indicated parasites with addition of 0, 0.3, 0.6, 1.8, 5, and 15 µM ISPA-28 (top to bottom traces in each panel, respectively). Inhibition in TFLC3 and miniSOG2 is intermediate between those observed for 3D7 and Dd2. (C) Ribbon diagrams showing expression of two CLAG3 isoforms in TFLC3 and miniSOG2 . (D) Immunofluorescence images showing trafficking of the 3D7-miniSOG-FLAG CLAG3 isoform in miniSOG2 . This protein, expressed under the msp2 promoter, colocalizes with RhopH3 in schizonts and is exported normally in trophozoites after invasion of new erythrocytes. Bar, 5 µm. (E and F) Immunoblot assays using membranes from miniSOG2 probed with anti-FLAG (E) and anti-CLAG3 (F) antibodies. Proteins were separated after cells were incubated without and with pronase E treatment (− and +, respectively). Both CLAG3 proteins expressed in this parasite are integral to the erythrocyte membrane. (G) ISPA-28 dose responses from experiments as in panel B. Symbols represent mean ± SEM for wild-type parasites (3D7, white circles; Dd2, black circles) and merodiploid transfectants ( TFLC3 , blue triangles; miniSOG2 , red triangles).
    Figure Legend Snippet: CLAG3 merodiploids reveal a stoichiometric contribution to PSAC-mediated nutrient uptake. (A) Schematic showing the minimum distinct combinations of host membrane RhopH complexes in the TFLC3 parasite. CLAG3 is shown as blue ribbon dimers embedded in the host membrane; note the permutations of the two C-terminal epitope tags (gold circles and pink ellipses). RhopH2 and RhopH3 are represented in green. (B) Sorbitol uptake kinetics for indicated parasites with addition of 0, 0.3, 0.6, 1.8, 5, and 15 µM ISPA-28 (top to bottom traces in each panel, respectively). Inhibition in TFLC3 and miniSOG2 is intermediate between those observed for 3D7 and Dd2. (C) Ribbon diagrams showing expression of two CLAG3 isoforms in TFLC3 and miniSOG2 . (D) Immunofluorescence images showing trafficking of the 3D7-miniSOG-FLAG CLAG3 isoform in miniSOG2 . This protein, expressed under the msp2 promoter, colocalizes with RhopH3 in schizonts and is exported normally in trophozoites after invasion of new erythrocytes. Bar, 5 µm. (E and F) Immunoblot assays using membranes from miniSOG2 probed with anti-FLAG (E) and anti-CLAG3 (F) antibodies. Proteins were separated after cells were incubated without and with pronase E treatment (− and +, respectively). Both CLAG3 proteins expressed in this parasite are integral to the erythrocyte membrane. (G) ISPA-28 dose responses from experiments as in panel B. Symbols represent mean ± SEM for wild-type parasites (3D7, white circles; Dd2, black circles) and merodiploid transfectants ( TFLC3 , blue triangles; miniSOG2 , red triangles).

    Techniques Used: Inhibition, Expressing, Immunofluorescence, Incubation

    16) Product Images from "RIPK1 protects hepatocytes from death in Fas-induced hepatitis"

    Article Title: RIPK1 protects hepatocytes from death in Fas-induced hepatitis

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-09789-8

    RIPK1 deficiency increases the sensitivity of primary hepatocytes to Fas-agonist stimulation. Primary cultures of hepatocytes issued from Ripk1 fl/fl ( n = 2) or Ripk1 LPC-KO (n = 2) mice were subjected during 16 h to mAb-Jo2 concentrations ranging from 1 to 5 ng/mL in presence of ETA (1 µg/mL) (left panel). Additional primary cultures (n = 3 for each strains) were exposed to a unique dose of mAb-Jo2 (5 ng/mL) also in presence of ETA (1 µg/mL) for statistical analysis (middle panel). In parallel, these primary hepatocyte cultures were subjected during 16 h to mAb-Jo2 at 20 ng/mL in presence of ETA (1 µg/mL) and in absence or presence of the z-VAD-fmk pan-caspase inhibitor (right panel). Cell death was analysed by WST-1 based assay and data are expressed as a percentage of signal obtained in basal survival conditions without Fas-agonist. Error bars corresponds either to internal triplicates for each primary cultures (left panel) or to triplicates of independent primary cultures (middle and right panels).
    Figure Legend Snippet: RIPK1 deficiency increases the sensitivity of primary hepatocytes to Fas-agonist stimulation. Primary cultures of hepatocytes issued from Ripk1 fl/fl ( n = 2) or Ripk1 LPC-KO (n = 2) mice were subjected during 16 h to mAb-Jo2 concentrations ranging from 1 to 5 ng/mL in presence of ETA (1 µg/mL) (left panel). Additional primary cultures (n = 3 for each strains) were exposed to a unique dose of mAb-Jo2 (5 ng/mL) also in presence of ETA (1 µg/mL) for statistical analysis (middle panel). In parallel, these primary hepatocyte cultures were subjected during 16 h to mAb-Jo2 at 20 ng/mL in presence of ETA (1 µg/mL) and in absence or presence of the z-VAD-fmk pan-caspase inhibitor (right panel). Cell death was analysed by WST-1 based assay and data are expressed as a percentage of signal obtained in basal survival conditions without Fas-agonist. Error bars corresponds either to internal triplicates for each primary cultures (left panel) or to triplicates of independent primary cultures (middle and right panels).

    Techniques Used: Mouse Assay, WST-1 Assay

    17) Product Images from "Emergence of a Large-Plaque Variant in Mice Infected with Coxsackievirus B3"

    Article Title: Emergence of a Large-Plaque Variant in Mice Infected with Coxsackievirus B3

    Journal: mBio

    doi: 10.1128/mBio.00119-16

    N63Y mutant CVB3 has a growth defect in cell culture and reduced glycan-mediated cell attachment. Single-cycle assays of viral replication in HeLa (A) and Huh7 (B) cells were performed. Infections with WT or N63Y mutant CVB3 were performed at an MOI of 0.1. Viral titers were determined by plaque assay with HeLa cells. n = 3. (C) Cell attachment of 35 S-labeled WT or N63Y mutant CVB3. Virus was incubated with cells at 4°C for 40 min. Cells were washed and trypsinized, and cell-associated 35 S was quantified. (D) 35 S-labeled WT or N63Y mutant CVB3 was incubated with CHO cells (CHO-K1, pgsA745, pgsD677, and pgsB761) that vary in GAG expression. Plus and minus signs indicate the relative levels of GAGs on the cell surface. (E) Effect of heparinase treatment on CVB3 cell attachment. Huh7 cells were treated with or without heparinase I for 90 min prior to quantification of 35 S CVB3 attachment. n = 7. (F) Heparin-agarose pulldown assay. 35 S-labeled WT or N63Y mutant CVB3 was incubated with heparin-agarose resin or streptavidin-agarose resin (control). Resin was washed, and bound 35 S-labeled CVB3 was quantified. n = 3. *, P
    Figure Legend Snippet: N63Y mutant CVB3 has a growth defect in cell culture and reduced glycan-mediated cell attachment. Single-cycle assays of viral replication in HeLa (A) and Huh7 (B) cells were performed. Infections with WT or N63Y mutant CVB3 were performed at an MOI of 0.1. Viral titers were determined by plaque assay with HeLa cells. n = 3. (C) Cell attachment of 35 S-labeled WT or N63Y mutant CVB3. Virus was incubated with cells at 4°C for 40 min. Cells were washed and trypsinized, and cell-associated 35 S was quantified. (D) 35 S-labeled WT or N63Y mutant CVB3 was incubated with CHO cells (CHO-K1, pgsA745, pgsD677, and pgsB761) that vary in GAG expression. Plus and minus signs indicate the relative levels of GAGs on the cell surface. (E) Effect of heparinase treatment on CVB3 cell attachment. Huh7 cells were treated with or without heparinase I for 90 min prior to quantification of 35 S CVB3 attachment. n = 7. (F) Heparin-agarose pulldown assay. 35 S-labeled WT or N63Y mutant CVB3 was incubated with heparin-agarose resin or streptavidin-agarose resin (control). Resin was washed, and bound 35 S-labeled CVB3 was quantified. n = 3. *, P

    Techniques Used: Mutagenesis, Cell Culture, Cell Attachment Assay, Plaque Assay, Labeling, Incubation, Expressing

    18) Product Images from "The episodic ataxia type 1 mutation I262T alters voltage-dependent gating and disrupts protein biosynthesis of human Kv1.1 potassium channels"

    Article Title: The episodic ataxia type 1 mutation I262T alters voltage-dependent gating and disrupts protein biosynthesis of human Kv1.1 potassium channels

    Journal: Scientific Reports

    doi: 10.1038/srep19378

    Biochemical analyses of the mechanism underlying the dominant-negative effect of I262T. ( A ) Surface biotinylation analyses of Myc-tagged Kv1.1 (Myc-Kv1.1) WT and I262T in HEK293T cells. ( Left ) Representative immunoblots. The molecular weight markers (in kilodaltons) are labeled to the left, and the immunoblotting antibodies (α-Myc and α-GAPDH) are specified below the immunoblots. Cell lysates from biotinylated intact cells were either directly employed for immunoblotting analyses (total) or subject to streptavidin pull-down before being used for immunoblotting analyses (surface). ( Right ) Quantification of total protein level (total signal), surface protein level (surface signal), and surface expression efficiency (surface/total signal). I262T shows reduced protein level. The total protein density was standardized as the ratio of total Myc signal to the signal of the loading control GAPDH. The surface protein density was standardized as the ratio of surface Myc signal to the cognate total GAPDH signal. The efficiency of surface presentation is expressed as surface protein density divided by the corresponding standardized total protein density. ( B ) The kinetics of Myc-Kv1.1-WT and Myc-Kv1.1-I262T protein degradation in the presence of 100 μg/ml cycloheximide (CHX) treatments of different durations. ( Left ) Representative immunoblots. ( Right ) Quantification of Kv1.1 protein degradation time course. Protein densities were standardized as the ratio of Kv1.1 signals to the cognate GAPDH signals, followed by normalization to those of the corresponding control at 0 hr. See Supplementary Fig. S3 for details on semi-logarithmic linear-regression analyses of the degradation time course. ( C ) Surface biotinylation analyses of Myc-Kv1.1-WT co-expressed with untagged WT or I262T (1:1 molar ratio). ( D ) Surface biotinylation analyses of Kv1.1 WT-WT dimer and WT-I262T dimer. Kv1.1 dimers were detected with the anti-Kv1.1 (αKv1.1) antibody. Asterisks denote significant difference from the WT control (*, t -test: p
    Figure Legend Snippet: Biochemical analyses of the mechanism underlying the dominant-negative effect of I262T. ( A ) Surface biotinylation analyses of Myc-tagged Kv1.1 (Myc-Kv1.1) WT and I262T in HEK293T cells. ( Left ) Representative immunoblots. The molecular weight markers (in kilodaltons) are labeled to the left, and the immunoblotting antibodies (α-Myc and α-GAPDH) are specified below the immunoblots. Cell lysates from biotinylated intact cells were either directly employed for immunoblotting analyses (total) or subject to streptavidin pull-down before being used for immunoblotting analyses (surface). ( Right ) Quantification of total protein level (total signal), surface protein level (surface signal), and surface expression efficiency (surface/total signal). I262T shows reduced protein level. The total protein density was standardized as the ratio of total Myc signal to the signal of the loading control GAPDH. The surface protein density was standardized as the ratio of surface Myc signal to the cognate total GAPDH signal. The efficiency of surface presentation is expressed as surface protein density divided by the corresponding standardized total protein density. ( B ) The kinetics of Myc-Kv1.1-WT and Myc-Kv1.1-I262T protein degradation in the presence of 100 μg/ml cycloheximide (CHX) treatments of different durations. ( Left ) Representative immunoblots. ( Right ) Quantification of Kv1.1 protein degradation time course. Protein densities were standardized as the ratio of Kv1.1 signals to the cognate GAPDH signals, followed by normalization to those of the corresponding control at 0 hr. See Supplementary Fig. S3 for details on semi-logarithmic linear-regression analyses of the degradation time course. ( C ) Surface biotinylation analyses of Myc-Kv1.1-WT co-expressed with untagged WT or I262T (1:1 molar ratio). ( D ) Surface biotinylation analyses of Kv1.1 WT-WT dimer and WT-I262T dimer. Kv1.1 dimers were detected with the anti-Kv1.1 (αKv1.1) antibody. Asterisks denote significant difference from the WT control (*, t -test: p

    Techniques Used: Dominant Negative Mutation, Western Blot, Molecular Weight, Labeling, Expressing

    19) Product Images from "Emergence of a Large-Plaque Variant in Mice Infected with Coxsackievirus B3"

    Article Title: Emergence of a Large-Plaque Variant in Mice Infected with Coxsackievirus B3

    Journal: mBio

    doi: 10.1128/mBio.00119-16

    The emergence of the large-plaque variant differs in various tissues. Four- to 6-week-old male IFNAR −/− or IFNAR +/+ mice were orally inoculated i.p. or i.m. with 5 × 10 7 PFU of WT CVB3. Fecal samples were collected from orally inoculated mice at 72 hpi. Liver, heart, and spleen samples were harvested from i.p. inoculated mice at 48 hpi. Muscle and liver samples were harvested from i.m. inoculated mice at 48 hpi. Following processing, viruses were plated on HeLa cells by using agar overlays. The sizes of 100 randomly picked plaques were quantified with ImageJ. The total percentages of large plaques among hundreds of plaques per condition are indicated at the top. Large plaques are defined as the average inoculum plaque size multiplied by a factor of 10 (2.473 mm 2 or larger; indicated by a dashed line).
    Figure Legend Snippet: The emergence of the large-plaque variant differs in various tissues. Four- to 6-week-old male IFNAR −/− or IFNAR +/+ mice were orally inoculated i.p. or i.m. with 5 × 10 7 PFU of WT CVB3. Fecal samples were collected from orally inoculated mice at 72 hpi. Liver, heart, and spleen samples were harvested from i.p. inoculated mice at 48 hpi. Muscle and liver samples were harvested from i.m. inoculated mice at 48 hpi. Following processing, viruses were plated on HeLa cells by using agar overlays. The sizes of 100 randomly picked plaques were quantified with ImageJ. The total percentages of large plaques among hundreds of plaques per condition are indicated at the top. Large plaques are defined as the average inoculum plaque size multiplied by a factor of 10 (2.473 mm 2 or larger; indicated by a dashed line).

    Techniques Used: Variant Assay, Mouse Assay

    VP3 N63Y is sufficient for the large-plaque phenotype. RT-PCR products from several large plaques were sequenced and found to contain the VP3 N63Y mutation. The N63Y mutant was cloned into a new infectious clone, and virus was generated. (A) Alignment of the VP3-63 region sequences of various viruses. The highlighted residue is in position 63. (B) CVB3 structure with the location of VP3-63 in red. The inset shows one 5-fold symmetry axis, with the VP3 proteins in gray. (C) Plaque phenotypes of WT CVB3, a plaque-purified fecal isolate with the large-plaque phenotype, and N63Y mutant CVB3.
    Figure Legend Snippet: VP3 N63Y is sufficient for the large-plaque phenotype. RT-PCR products from several large plaques were sequenced and found to contain the VP3 N63Y mutation. The N63Y mutant was cloned into a new infectious clone, and virus was generated. (A) Alignment of the VP3-63 region sequences of various viruses. The highlighted residue is in position 63. (B) CVB3 structure with the location of VP3-63 in red. The inset shows one 5-fold symmetry axis, with the VP3 proteins in gray. (C) Plaque phenotypes of WT CVB3, a plaque-purified fecal isolate with the large-plaque phenotype, and N63Y mutant CVB3.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Mutagenesis, Clone Assay, Generated, Purification

    N63Y mutant CVB3 shows greater dissemination to the liver and induces more liver damage than WT CVB3. IFNAR −/− mice were orally infected with 5 × 10 7 PFU of WT or N63Y mutant CVB3. (A) Blood was harvested at 24 and 72 hpi, and ALT levels were quantified by ELISA. n = 6. (B) IFNAR −/− mice were inoculated i.p. with 2 × 10 7 PFU/20,000 cpm 35 S labeled WT or N63Y mutant CVB3. Heart, liver, and spleen samples were harvested at 1 hpi, and tissue-associated counts per minute were quantified. (C) Binding of CVB3 to homogenized tissues. A total of 3 × 10 6 PFU/3,000 cpm 35 S labeled WT or N63Y mutant CVB3 were incubated with homogenized liver tissue from IFNAR −/− mice for 60 min; this was followed by washing and quantification of the tissue-associated counts per minute. *, P
    Figure Legend Snippet: N63Y mutant CVB3 shows greater dissemination to the liver and induces more liver damage than WT CVB3. IFNAR −/− mice were orally infected with 5 × 10 7 PFU of WT or N63Y mutant CVB3. (A) Blood was harvested at 24 and 72 hpi, and ALT levels were quantified by ELISA. n = 6. (B) IFNAR −/− mice were inoculated i.p. with 2 × 10 7 PFU/20,000 cpm 35 S labeled WT or N63Y mutant CVB3. Heart, liver, and spleen samples were harvested at 1 hpi, and tissue-associated counts per minute were quantified. (C) Binding of CVB3 to homogenized tissues. A total of 3 × 10 6 PFU/3,000 cpm 35 S labeled WT or N63Y mutant CVB3 were incubated with homogenized liver tissue from IFNAR −/− mice for 60 min; this was followed by washing and quantification of the tissue-associated counts per minute. *, P

    Techniques Used: Mutagenesis, Mouse Assay, Infection, Enzyme-linked Immunosorbent Assay, Labeling, Binding Assay, Incubation

    N63Y mutant CVB3 has a growth defect in cell culture and reduced glycan-mediated cell attachment. Single-cycle assays of viral replication in HeLa (A) and Huh7 (B) cells were performed. Infections with WT or N63Y mutant CVB3 were performed at an MOI of 0.1. Viral titers were determined by plaque assay with HeLa cells. n = 3. (C) Cell attachment of 35 S-labeled WT or N63Y mutant CVB3. Virus was incubated with cells at 4°C for 40 min. Cells were washed and trypsinized, and cell-associated 35 S was quantified. (D) 35 S-labeled WT or N63Y mutant CVB3 was incubated with CHO cells (CHO-K1, pgsA745, pgsD677, and pgsB761) that vary in GAG expression. Plus and minus signs indicate the relative levels of GAGs on the cell surface. (E) Effect of heparinase treatment on CVB3 cell attachment. Huh7 cells were treated with or without heparinase I for 90 min prior to quantification of 35 S CVB3 attachment. n = 7. (F) Heparin-agarose pulldown assay. 35 S-labeled WT or N63Y mutant CVB3 was incubated with heparin-agarose resin or streptavidin-agarose resin (control). Resin was washed, and bound 35 S-labeled CVB3 was quantified. n = 3. *, P
    Figure Legend Snippet: N63Y mutant CVB3 has a growth defect in cell culture and reduced glycan-mediated cell attachment. Single-cycle assays of viral replication in HeLa (A) and Huh7 (B) cells were performed. Infections with WT or N63Y mutant CVB3 were performed at an MOI of 0.1. Viral titers were determined by plaque assay with HeLa cells. n = 3. (C) Cell attachment of 35 S-labeled WT or N63Y mutant CVB3. Virus was incubated with cells at 4°C for 40 min. Cells were washed and trypsinized, and cell-associated 35 S was quantified. (D) 35 S-labeled WT or N63Y mutant CVB3 was incubated with CHO cells (CHO-K1, pgsA745, pgsD677, and pgsB761) that vary in GAG expression. Plus and minus signs indicate the relative levels of GAGs on the cell surface. (E) Effect of heparinase treatment on CVB3 cell attachment. Huh7 cells were treated with or without heparinase I for 90 min prior to quantification of 35 S CVB3 attachment. n = 7. (F) Heparin-agarose pulldown assay. 35 S-labeled WT or N63Y mutant CVB3 was incubated with heparin-agarose resin or streptavidin-agarose resin (control). Resin was washed, and bound 35 S-labeled CVB3 was quantified. n = 3. *, P

    Techniques Used: Mutagenesis, Cell Culture, Cell Attachment Assay, Plaque Assay, Labeling, Incubation, Expressing

    N63Y mutant CVB3 shows enhanced replication and virulence in mice. The viral fecal shedding profiles of IFNAR −/− (A) or IFNAR +/+ (B) mice orally inoculated with 5 × 10 7 PFU of WT or N63Y mutant CVB3 were determined. Virus titers were determined by plaque assay. Tissue viral titers at 72 hpi were determined in IFNAR −/− (C) or IFNAR +/+ (D) mice orally inoculated with 5 × 10 7 PFU of WT or N63Y mutant CVB3. (E) Survival curves of CVB3-infected mice. From left to right, IFNAR −/− (blue lines) or IFNAR +/+ (gray lines) mice were inoculated orally with 5 × 10 7 PFU, i.p. inoculated with 1 × 10 4 PFU, or i.m. inoculated with 1 × 10 2 PFU of WT (solid line) or N63Y mutant (dashed line) CVB3. For all panels, n = 5 to 8. *, P
    Figure Legend Snippet: N63Y mutant CVB3 shows enhanced replication and virulence in mice. The viral fecal shedding profiles of IFNAR −/− (A) or IFNAR +/+ (B) mice orally inoculated with 5 × 10 7 PFU of WT or N63Y mutant CVB3 were determined. Virus titers were determined by plaque assay. Tissue viral titers at 72 hpi were determined in IFNAR −/− (C) or IFNAR +/+ (D) mice orally inoculated with 5 × 10 7 PFU of WT or N63Y mutant CVB3. (E) Survival curves of CVB3-infected mice. From left to right, IFNAR −/− (blue lines) or IFNAR +/+ (gray lines) mice were inoculated orally with 5 × 10 7 PFU, i.p. inoculated with 1 × 10 4 PFU, or i.m. inoculated with 1 × 10 2 PFU of WT (solid line) or N63Y mutant (dashed line) CVB3. For all panels, n = 5 to 8. *, P

    Techniques Used: Mutagenesis, Mouse Assay, Plaque Assay, Infection

    Emergence of a CVB3 large-plaque variant following oral inoculation. Four- to 6-week-old male IFNAR −/− mice were orally inoculated with 5 × 10 7 PFU of light-sensitive, neutral-red-labeled WT CVB3 in the dark, and fecal samples were collected at 24, 48, and 72 hpi in the dark. (A) Total viral titers determined by HeLa cell plaque assays with agar overlays. (B) Percentages of replicated (noninoculum) virus in fecal samples. Flowthrough inoculum viruses are light sensitive, whereas viruses that replicated in mice are light insensitive. Aliquots of each processed fecal sample were exposed to light or dark, and replication status was determined by dividing the number of PFU/ml of light-exposed samples by the number of PFU/ml of dark-exposed samples and multiplying by 100%. Each symbol represents one mouse. (C) Representative plaque morphologies of inoculum and 48- and 72-hpi fecal samples. (D) Plaque size quantification. Each symbol represents a plaque. Large plaques are defined as the average inoculum plaque size multiplied by a factor of 10 (2.473 mm 2 or larger; indicated by a dashed line). For panels A and B, n = 5 to 10.
    Figure Legend Snippet: Emergence of a CVB3 large-plaque variant following oral inoculation. Four- to 6-week-old male IFNAR −/− mice were orally inoculated with 5 × 10 7 PFU of light-sensitive, neutral-red-labeled WT CVB3 in the dark, and fecal samples were collected at 24, 48, and 72 hpi in the dark. (A) Total viral titers determined by HeLa cell plaque assays with agar overlays. (B) Percentages of replicated (noninoculum) virus in fecal samples. Flowthrough inoculum viruses are light sensitive, whereas viruses that replicated in mice are light insensitive. Aliquots of each processed fecal sample were exposed to light or dark, and replication status was determined by dividing the number of PFU/ml of light-exposed samples by the number of PFU/ml of dark-exposed samples and multiplying by 100%. Each symbol represents one mouse. (C) Representative plaque morphologies of inoculum and 48- and 72-hpi fecal samples. (D) Plaque size quantification. Each symbol represents a plaque. Large plaques are defined as the average inoculum plaque size multiplied by a factor of 10 (2.473 mm 2 or larger; indicated by a dashed line). For panels A and B, n = 5 to 10.

    Techniques Used: Variant Assay, Mouse Assay, Labeling

    20) Product Images from "Botulinum Neurotoxin Serotype A Recognizes Its Protein Receptor SV2 by a Different Mechanism than Botulinum Neurotoxin B Synaptotagmin"

    Article Title: Botulinum Neurotoxin Serotype A Recognizes Its Protein Receptor SV2 by a Different Mechanism than Botulinum Neurotoxin B Synaptotagmin

    Journal: Toxins

    doi: 10.3390/toxins8050154

    CD secondary structure analyses of free and bound hSV2C peptide. ( A ) GST-hSV2C 455–579 was quantitatively digested with thrombin and the released hSV2C peptide (15.6 kDa) was isolated by gelfiltration (fractions 11a–15). ( B ) Far-UV CD spectra of free hSV2C peptide (5 µM; red trace), free H C AS (8 µM, dark blue) and a 1:1 mixture of hSV2C-H C AS (5 µM; black). Arithmetic addition of spectra of free hSV2C (red) and free H C AS (dark blue) yields a spectrum (grey) similar to that of the hSV2C-H C AS mixture (black). Accordingly, subtraction of CD signal of either hSV2C (light blue) or H C AS (orange) from that of the hSV2C-H C AS mixture yielded spectra similar to that of the corresponding free protein.
    Figure Legend Snippet: CD secondary structure analyses of free and bound hSV2C peptide. ( A ) GST-hSV2C 455–579 was quantitatively digested with thrombin and the released hSV2C peptide (15.6 kDa) was isolated by gelfiltration (fractions 11a–15). ( B ) Far-UV CD spectra of free hSV2C peptide (5 µM; red trace), free H C AS (8 µM, dark blue) and a 1:1 mixture of hSV2C-H C AS (5 µM; black). Arithmetic addition of spectra of free hSV2C (red) and free H C AS (dark blue) yields a spectrum (grey) similar to that of the hSV2C-H C AS mixture (black). Accordingly, subtraction of CD signal of either hSV2C (light blue) or H C AS (orange) from that of the hSV2C-H C AS mixture yielded spectra similar to that of the corresponding free protein.

    Techniques Used: Isolation

    SPR-sensorgrams of the interaction of recombinant H C B with: GST–rSyt-II 1–61 ( A ); recombinant H C A with GST-rSV2C 454–579 ( B ); GST-hSV2C 455–579 ( C ); GST-hSV2C 455–579 at pH 5.0 ( D ); or GST-hSV2C 455–579 F563L ( E ) (Inserts: Fits for determination of steady-state affinity). The measured double referenced binding responses (ΔRU; red lines) are overlaid with fits of a 1:1 Langmuir interaction (black lines). For measurements of GST-rSV2C at pH 7.3 ( B ) and GST-hSV2C at pH 5.0 ( D ), kinetic evaluation was omitted due to heterogeneity in the binding curves (( B ) dashed lines: fit for 1:1 Langmuir interaction shows clear deviations) or too low binding responses (( D ) no fit shown). H C A and H C B were injected in three-fold dilution series starting from 1200 nM down to 14.8 nM. The highest H C concentration was injected in duplicates except for GST-rSV2C 454–579 and GST-hSV2C at pH 5.0.
    Figure Legend Snippet: SPR-sensorgrams of the interaction of recombinant H C B with: GST–rSyt-II 1–61 ( A ); recombinant H C A with GST-rSV2C 454–579 ( B ); GST-hSV2C 455–579 ( C ); GST-hSV2C 455–579 at pH 5.0 ( D ); or GST-hSV2C 455–579 F563L ( E ) (Inserts: Fits for determination of steady-state affinity). The measured double referenced binding responses (ΔRU; red lines) are overlaid with fits of a 1:1 Langmuir interaction (black lines). For measurements of GST-rSV2C at pH 7.3 ( B ) and GST-hSV2C at pH 5.0 ( D ), kinetic evaluation was omitted due to heterogeneity in the binding curves (( B ) dashed lines: fit for 1:1 Langmuir interaction shows clear deviations) or too low binding responses (( D ) no fit shown). H C A and H C B were injected in three-fold dilution series starting from 1200 nM down to 14.8 nM. The highest H C concentration was injected in duplicates except for GST-rSV2C 454–579 and GST-hSV2C at pH 5.0.

    Techniques Used: SPR Assay, Recombinant, Binding Assay, Injection, Concentration Assay

    ( A ) Determination of binding constant K D of single chain H6tBoNTA to rSV2C 454–579: GST-rSV2C 454–579 (75 pmol) was immobilized and 31 nM–1 µM single-chain H6tBoNTA in 100 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.5% Triton X-100 were added as ligand ( n = 2). Bound H6tBoNTA was quantified by SDS-PAGE and densitometry upon Coomassie blue staining. ( B ) Comparison of SV2C-LD4 fused to different affinity tags in pull down experiments. GST-rSV2C 454–579, GST-hSV2C 455–579, GST-hSV2C 455–579 F563L, H6hSV2C6xHN and hSV2C6xHN (each 75 pmol) were immobilized and tag-free di-chain BoNTA in PBS, 0.5% Triton X-100, pH 7.4 was added in ten different concentrations (15–7600 nM) as ligand ( n = 3); ( C ) Representative SDS-PAGE analysis of a GST pull down experiment employing immobilized GST-hSV2C 455–579 F563L and tag-free di-chain BoNTA (15–4000 nM).
    Figure Legend Snippet: ( A ) Determination of binding constant K D of single chain H6tBoNTA to rSV2C 454–579: GST-rSV2C 454–579 (75 pmol) was immobilized and 31 nM–1 µM single-chain H6tBoNTA in 100 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.5% Triton X-100 were added as ligand ( n = 2). Bound H6tBoNTA was quantified by SDS-PAGE and densitometry upon Coomassie blue staining. ( B ) Comparison of SV2C-LD4 fused to different affinity tags in pull down experiments. GST-rSV2C 454–579, GST-hSV2C 455–579, GST-hSV2C 455–579 F563L, H6hSV2C6xHN and hSV2C6xHN (each 75 pmol) were immobilized and tag-free di-chain BoNTA in PBS, 0.5% Triton X-100, pH 7.4 was added in ten different concentrations (15–7600 nM) as ligand ( n = 3); ( C ) Representative SDS-PAGE analysis of a GST pull down experiment employing immobilized GST-hSV2C 455–579 F563L and tag-free di-chain BoNTA (15–4000 nM).

    Techniques Used: Binding Assay, SDS Page, Staining

    GST pull down experiments employing GST-rSV2A 468–594, GST-rSV2B 413–535, GST-rSV2C 454–579 and GST-hSV2C 455–579 (each 150 pmol) without complex gangliosides and H C AS wt (500 nM) as ligand in 20 mM Tris-HCl pH 7.4, 80 mM NaCl, 0.5% Triton X-100. Mean ± SD of H C AS absolute binding in mol % to rSV2A ( n = 3), rSV2B ( n = 3), rSV2C ( n = 14) and hSV2C ( n = 9) after subtraction of background binding to GST-GT matrix is shown.
    Figure Legend Snippet: GST pull down experiments employing GST-rSV2A 468–594, GST-rSV2B 413–535, GST-rSV2C 454–579 and GST-hSV2C 455–579 (each 150 pmol) without complex gangliosides and H C AS wt (500 nM) as ligand in 20 mM Tris-HCl pH 7.4, 80 mM NaCl, 0.5% Triton X-100. Mean ± SD of H C AS absolute binding in mol % to rSV2A ( n = 3), rSV2B ( n = 3), rSV2C ( n = 14) and hSV2C ( n = 9) after subtraction of background binding to GST-GT matrix is shown.

    Techniques Used: Binding Assay

    21) Product Images from "Amyloid-like aggregation of provasopressin in diabetes insipidus and secretory granule sorting"

    Article Title: Amyloid-like aggregation of provasopressin in diabetes insipidus and secretory granule sorting

    Journal: BMC Biology

    doi: 10.1186/s12915-017-0347-9

    Stimulated secretion depends on both vasopressin and glycopeptide. a AtT20 cells stably expressing wild-type provasopressin ( wt ) or the mutants without the glycopeptide (∆ gp ) or with the V5xA mutant hormone sequence ( 5xA ) or both ( 5xA∆gp ), or expressing the constitutive secretory protein A1Pi were grown and incubated for 30 min each with serum-free medium without (−) and then with BaCl 2 (+) to stimulate granule secretion for 30 min each. The media were collected, concentrated, and analyzed by SDS-gel electrophoresis and immunoblotting. To keep intensities within the diagnostic range of immunoblot analysis, a threefold larger aliquot (3x) of the control medium was analyzed than of the stimulation medium (1x). b Immunoblots as in A were quantified and stimulated secretion plotted as the ratio of stimulated to constitutive secretion ( s/c ). The mean and the individual values of four to five independent experiments are shown. Except for wild-type vs. V5xA, all pairwise comparisons are significantly different according to an unpaired two-tailed t test (the most important ones are indicated above the graph; * p
    Figure Legend Snippet: Stimulated secretion depends on both vasopressin and glycopeptide. a AtT20 cells stably expressing wild-type provasopressin ( wt ) or the mutants without the glycopeptide (∆ gp ) or with the V5xA mutant hormone sequence ( 5xA ) or both ( 5xA∆gp ), or expressing the constitutive secretory protein A1Pi were grown and incubated for 30 min each with serum-free medium without (−) and then with BaCl 2 (+) to stimulate granule secretion for 30 min each. The media were collected, concentrated, and analyzed by SDS-gel electrophoresis and immunoblotting. To keep intensities within the diagnostic range of immunoblot analysis, a threefold larger aliquot (3x) of the control medium was analyzed than of the stimulation medium (1x). b Immunoblots as in A were quantified and stimulated secretion plotted as the ratio of stimulated to constitutive secretion ( s/c ). The mean and the individual values of four to five independent experiments are shown. Except for wild-type vs. V5xA, all pairwise comparisons are significantly different according to an unpaired two-tailed t test (the most important ones are indicated above the graph; * p

    Techniques Used: Stable Transfection, Expressing, Mutagenesis, Sequencing, Incubation, SDS-Gel, Electrophoresis, Diagnostic Assay, Western Blot, Two Tailed Test

    Granule sorting is mediated by both vasopressin and glycopeptide. a Stable AtT20 cells expressing the indicated provasopressin constructs were labeled with [ 35 S]methionine for 30 min and chased for an additional 2 h. From cell lysates and media, products were immunoprecipitated and analyzed by gel electrophoresis and autoradiography. The positions of full-size glycosylated ( FLg ) and unglycosylated forms ( FLu ) and of constructs without glycopeptide (∆ gp ) are indicated. b – e AtT20 cells stably expressing wild-type provasopressin ( wt ) or the mutants without the glycopeptide (∆ gp ) or with the V5xA mutant hormone sequence ( 5xA ) or both ( 5xA∆gp ) were stained for CgA as a granule marker, NPII, and nuclei ( blue ). Granules concentrate in terminal patches. Bar : 10 μm. f Quantitation of NPII staining in granule patches normalized for content of CgA. NPII/CgA ratios of ~70 images per construct (45 images for untransfected AtT20 cells, − ) containing 4–5 cells each are summarized as a box plot showing the median and the center 50% of values in the box , with the whiskers including the 10th to the 90th percentiles. The central dot represents the mean. The original quantitation is shown on the left in gray and white . In black and gray , the data are shown upon correction for the relative amounts of pulse-labeled protein recovered from both media and cell lysates after a 2-h chase (three independent experiments including that shown in panel a ; wt, 1.0; V5xA, 0.77 ± 0.20; ∆gp, 0.78 ± 0.20; V5xA∆gp, 0.68 ± 0.19). All pairwise comparisons, except wild-type vs. V∆gp, are significantly different according to an unpaired two-tailed t test ( p
    Figure Legend Snippet: Granule sorting is mediated by both vasopressin and glycopeptide. a Stable AtT20 cells expressing the indicated provasopressin constructs were labeled with [ 35 S]methionine for 30 min and chased for an additional 2 h. From cell lysates and media, products were immunoprecipitated and analyzed by gel electrophoresis and autoradiography. The positions of full-size glycosylated ( FLg ) and unglycosylated forms ( FLu ) and of constructs without glycopeptide (∆ gp ) are indicated. b – e AtT20 cells stably expressing wild-type provasopressin ( wt ) or the mutants without the glycopeptide (∆ gp ) or with the V5xA mutant hormone sequence ( 5xA ) or both ( 5xA∆gp ) were stained for CgA as a granule marker, NPII, and nuclei ( blue ). Granules concentrate in terminal patches. Bar : 10 μm. f Quantitation of NPII staining in granule patches normalized for content of CgA. NPII/CgA ratios of ~70 images per construct (45 images for untransfected AtT20 cells, − ) containing 4–5 cells each are summarized as a box plot showing the median and the center 50% of values in the box , with the whiskers including the 10th to the 90th percentiles. The central dot represents the mean. The original quantitation is shown on the left in gray and white . In black and gray , the data are shown upon correction for the relative amounts of pulse-labeled protein recovered from both media and cell lysates after a 2-h chase (three independent experiments including that shown in panel a ; wt, 1.0; V5xA, 0.77 ± 0.20; ∆gp, 0.78 ± 0.20; V5xA∆gp, 0.68 ± 0.19). All pairwise comparisons, except wild-type vs. V∆gp, are significantly different according to an unpaired two-tailed t test ( p

    Techniques Used: Expressing, Construct, Labeling, Immunoprecipitation, Nucleic Acid Electrophoresis, Autoradiography, Stable Transfection, Mutagenesis, Sequencing, Staining, Marker, Quantitation Assay, Two Tailed Test

    Pro/Gly scan through full-length provasopressin suggests more than one ER aggregating segment. a Domain organization of preprovasopressin. Cysteines are indicated by red dots , disulfide bonds by red lines , and glycosylation by a black diamond. Dots above the sequence indicate distinct mutations causing autosomal dominant neurohypophyseal DI (missense or deletion in black , nonsense or frameshift in pink ). The natural mutants used in this study, ∆E47 and C61X, are labeled. The scale indicates the number of the amino acids in provasopressin. In constructs Pro1–Pro10 successive segments of 10 residues in provasopressin were replaced by proline/glycine-rich sequences as illustrated. b The constructs were expressed in HN10 cells for 2 days and analyzed by immunofluorescence staining. Pro1/2/7/9-expressing cells are shown as examples. Nuclei were stained with DAPI ( blue ). Bar : 10 μm. c The fraction of expressing cells with ER aggregates was quantified and plotted (mean and individual values of three or four independent transfections (as indicated), analyzing ~200 expressing cells per transfection as described in “ Methods ”). d Immunoblot analysis of transfected cells after separation by reducing sodium dodecyl sulfate ( SDS )-gel electrophoresis is shown. Considerable amounts of SDS- and dithiothreitol-resistant provasopressin oligomers were detected for all proteins except Pro1. Molecular weight standards are indicated in kilodaltons
    Figure Legend Snippet: Pro/Gly scan through full-length provasopressin suggests more than one ER aggregating segment. a Domain organization of preprovasopressin. Cysteines are indicated by red dots , disulfide bonds by red lines , and glycosylation by a black diamond. Dots above the sequence indicate distinct mutations causing autosomal dominant neurohypophyseal DI (missense or deletion in black , nonsense or frameshift in pink ). The natural mutants used in this study, ∆E47 and C61X, are labeled. The scale indicates the number of the amino acids in provasopressin. In constructs Pro1–Pro10 successive segments of 10 residues in provasopressin were replaced by proline/glycine-rich sequences as illustrated. b The constructs were expressed in HN10 cells for 2 days and analyzed by immunofluorescence staining. Pro1/2/7/9-expressing cells are shown as examples. Nuclei were stained with DAPI ( blue ). Bar : 10 μm. c The fraction of expressing cells with ER aggregates was quantified and plotted (mean and individual values of three or four independent transfections (as indicated), analyzing ~200 expressing cells per transfection as described in “ Methods ”). d Immunoblot analysis of transfected cells after separation by reducing sodium dodecyl sulfate ( SDS )-gel electrophoresis is shown. Considerable amounts of SDS- and dithiothreitol-resistant provasopressin oligomers were detected for all proteins except Pro1. Molecular weight standards are indicated in kilodaltons

    Techniques Used: Sequencing, Labeling, Construct, Immunofluorescence, Staining, Expressing, Transfection, SDS-Gel, Electrophoresis, Molecular Weight

    ER aggregation of C-terminally truncated forms of provasopressin. a Schematic representation of C-terminally truncated provasopressin mutants C61myc (corresponding to 1-72myc), 1-60myc, and 1-50myc. The myc epitope is shown in purple . b These constructs were expressed in HN10 cells and visualized by immunofluorescence microscopy. Cells expressing C61myc were costained for the ER chaperone calreticulin. Nuclei were stained with DAPI ( blue ). Bar : 10 μm. c The fraction of expressing cells with ER aggregates was quantified and plotted (mean and individual values of four independent transfections, analyzing ~200 expressing cells per transfection). d Immunoblot analysis of untransfected cells ( − ) and transfected cells expressing C61myc after reducing SDS-gel electrophoresis. Molecular weight standards are indicated in kilodaltons. e Electron micrographs of C61myc aggregates forming a fibrillar network decorated with 10-nm gold. The surrounding rough ER membrane is visible in the left micrograph. Bars : 200 nm. Below , a section is shown enlarged with and without fibrillar lines highlighted for clarity
    Figure Legend Snippet: ER aggregation of C-terminally truncated forms of provasopressin. a Schematic representation of C-terminally truncated provasopressin mutants C61myc (corresponding to 1-72myc), 1-60myc, and 1-50myc. The myc epitope is shown in purple . b These constructs were expressed in HN10 cells and visualized by immunofluorescence microscopy. Cells expressing C61myc were costained for the ER chaperone calreticulin. Nuclei were stained with DAPI ( blue ). Bar : 10 μm. c The fraction of expressing cells with ER aggregates was quantified and plotted (mean and individual values of four independent transfections, analyzing ~200 expressing cells per transfection). d Immunoblot analysis of untransfected cells ( − ) and transfected cells expressing C61myc after reducing SDS-gel electrophoresis. Molecular weight standards are indicated in kilodaltons. e Electron micrographs of C61myc aggregates forming a fibrillar network decorated with 10-nm gold. The surrounding rough ER membrane is visible in the left micrograph. Bars : 200 nm. Below , a section is shown enlarged with and without fibrillar lines highlighted for clarity

    Techniques Used: Construct, Immunofluorescence, Microscopy, Expressing, Staining, Transfection, SDS-Gel, Electrophoresis, Molecular Weight

    The glycopeptide sequence contains a second aggregation sequence. a Full-size provasopressin and deletion mutants lacking the glycopeptide (∆ gp ) or the C-terminal half of the protein (1–75), with or without the ∆E47 point mutation or the Pro1, Pro2, or Pro3 replacement (as indicated) were constructed to localize the second aggregation sequence. The truncated proteins were provided with a His 6 tag ( black bar ). b Constructs were expressed in HN10 cells and analyzed by immunofluorescence as shown for selected examples. Bar : 10 μm. c The fraction of expressing cells forming ER aggregates was quantified as before (mean and individual values of three or four independent transfections (as indicated), analyzing ~200 expressing cells per transfection). For comparison, values for Pro1/2/3, 1–75, and 1–75Pro1/2/3 from Figs. 1c and 3c , respectively, are also shown. d , e Cells expressing construct Pro1, which lacks the N-terminal aggregation sequence, were analyzed by electron microscopy with immunogold labeling. Aggregates formed via the glycopeptide sequence showed different degrees of compaction (see also Additional file 2 : Figure S2). Some aggregates contained dense and light regions ( d ), while others were almost completely dense ( e ). Bars : 500 nm. Enlargements of the boxed regions below reveal a fibrillar network within the aggregates
    Figure Legend Snippet: The glycopeptide sequence contains a second aggregation sequence. a Full-size provasopressin and deletion mutants lacking the glycopeptide (∆ gp ) or the C-terminal half of the protein (1–75), with or without the ∆E47 point mutation or the Pro1, Pro2, or Pro3 replacement (as indicated) were constructed to localize the second aggregation sequence. The truncated proteins were provided with a His 6 tag ( black bar ). b Constructs were expressed in HN10 cells and analyzed by immunofluorescence as shown for selected examples. Bar : 10 μm. c The fraction of expressing cells forming ER aggregates was quantified as before (mean and individual values of three or four independent transfections (as indicated), analyzing ~200 expressing cells per transfection). For comparison, values for Pro1/2/3, 1–75, and 1–75Pro1/2/3 from Figs. 1c and 3c , respectively, are also shown. d , e Cells expressing construct Pro1, which lacks the N-terminal aggregation sequence, were analyzed by electron microscopy with immunogold labeling. Aggregates formed via the glycopeptide sequence showed different degrees of compaction (see also Additional file 2 : Figure S2). Some aggregates contained dense and light regions ( d ), while others were almost completely dense ( e ). Bars : 500 nm. Enlargements of the boxed regions below reveal a fibrillar network within the aggregates

    Techniques Used: Sequencing, Mutagenesis, Construct, Immunofluorescence, Expressing, Transfection, Electron Microscopy, Labeling

    The vasopressin nonapeptide is necessary for ER aggregation of provasopressin 1–75 and sufficient to aggregate a reporter construct. a Schematic presentation of provasopressin 1–75 (with a C-terminal His 6 tag in black ) and its proline/glycine scanning mutants 1–75Pro1–7. b Immunofluorescence localization of example constructs expressed in HN10 cells. The indicated constructs fused to a His 6 tag were transfected into HN10 cells and fluorescently stained using an anti-His 6 antibody. Bar : 10 μm. c Aggregation frequency of these constructs was quantified as before (mean and individual values of four independent transfections, analyzing ~200 expressing cells per transfection). d Transfected cells were lysed, separated by reducing SDS-gel electrophoresis, and subjected to immunoblot analysis to detect intracellularly accumulated provasopressin constructs. Molecular weight standards are indicated in kilodaltons. e Truncated provasopressin sequences 1–75 (positive control), 1–75Pro1 (negative control), 1–50, 1–25, 1–16, and 1–10 were fused to a reporter sequence ( Rep ) consisting of the C-terminal fragment 101–218 of glutathione S-transferase ( GST , orange ) with a myc epitope (in purple ). f Immunofluorescence localization of indicated constructs expressed in HN10 cells. Bar : 10 μm. g Aggregation frequency of the constructs shown in panel e was quantified as before (mean and individual values of three independent transfections for 1–75-Rep, 1–75Pro1-Rep, and 1–10-Rep, and single determinations for the others, analyzing ~100 expressing cells per transfection)
    Figure Legend Snippet: The vasopressin nonapeptide is necessary for ER aggregation of provasopressin 1–75 and sufficient to aggregate a reporter construct. a Schematic presentation of provasopressin 1–75 (with a C-terminal His 6 tag in black ) and its proline/glycine scanning mutants 1–75Pro1–7. b Immunofluorescence localization of example constructs expressed in HN10 cells. The indicated constructs fused to a His 6 tag were transfected into HN10 cells and fluorescently stained using an anti-His 6 antibody. Bar : 10 μm. c Aggregation frequency of these constructs was quantified as before (mean and individual values of four independent transfections, analyzing ~200 expressing cells per transfection). d Transfected cells were lysed, separated by reducing SDS-gel electrophoresis, and subjected to immunoblot analysis to detect intracellularly accumulated provasopressin constructs. Molecular weight standards are indicated in kilodaltons. e Truncated provasopressin sequences 1–75 (positive control), 1–75Pro1 (negative control), 1–50, 1–25, 1–16, and 1–10 were fused to a reporter sequence ( Rep ) consisting of the C-terminal fragment 101–218 of glutathione S-transferase ( GST , orange ) with a myc epitope (in purple ). f Immunofluorescence localization of indicated constructs expressed in HN10 cells. Bar : 10 μm. g Aggregation frequency of the constructs shown in panel e was quantified as before (mean and individual values of three independent transfections for 1–75-Rep, 1–75Pro1-Rep, and 1–10-Rep, and single determinations for the others, analyzing ~100 expressing cells per transfection)

    Techniques Used: Construct, Immunofluorescence, Transfection, Staining, Expressing, SDS-Gel, Electrophoresis, Molecular Weight, Positive Control, Negative Control, Sequencing

    22) Product Images from "Viral attachment induces rapid recruitment of an innate immune sensor TRIM5? to the plasma membrane"

    Article Title: Viral attachment induces rapid recruitment of an innate immune sensor TRIM5? to the plasma membrane

    Journal: Journal of innate immunity

    doi: 10.1159/000346963

    ]. Representative images from 3 independent experiments are shown. (A) FRhK4 cells stably expressing HA-tagged TRIM5αrh protein (FRhK4T5αHA) treated with PBS (upper panel) or HIV-1 VLPs (lower panel) are shown. Flotillin-1 was used as a marker for flotation fractions, while Nup98 was used as a sedimenting control. Antibody against HIV-1 Capsid (CA) is shown, p24 is indicated by arrow. (B) Identical experiments were carried out as A , however using SIV VLPs or BSA-coated nanoparticles. (C) Identical fractionation experiments were carried out as A , however using wild-type FRhK4 proteins expressing endogenous levels of TRIM5αrh. Endogenous TRIM5αrh was probed using a commercially-available antibody against TRIM5 (ProSci Incorporated).
    Figure Legend Snippet: ]. Representative images from 3 independent experiments are shown. (A) FRhK4 cells stably expressing HA-tagged TRIM5αrh protein (FRhK4T5αHA) treated with PBS (upper panel) or HIV-1 VLPs (lower panel) are shown. Flotillin-1 was used as a marker for flotation fractions, while Nup98 was used as a sedimenting control. Antibody against HIV-1 Capsid (CA) is shown, p24 is indicated by arrow. (B) Identical experiments were carried out as A , however using SIV VLPs or BSA-coated nanoparticles. (C) Identical fractionation experiments were carried out as A , however using wild-type FRhK4 proteins expressing endogenous levels of TRIM5αrh. Endogenous TRIM5αrh was probed using a commercially-available antibody against TRIM5 (ProSci Incorporated).

    Techniques Used: Stable Transfection, Expressing, Marker, Fractionation

    23) Product Images from "Sex-Differences in Renal Expression of Selected Transporters and Transcription Factors in Lean and Obese Zucker Spontaneously Hypertensive Fatty Rats"

    Article Title: Sex-Differences in Renal Expression of Selected Transporters and Transcription Factors in Lean and Obese Zucker Spontaneously Hypertensive Fatty Rats

    Journal: Journal of Diabetes Research

    doi: 10.1155/2015/483238

    IC 50 determination for the inhibition of OAT2-mediated cGMP uptake by furosemide and bumetanide. In HEK293 cells stably transfected with OAT2 or empty vector, intracellular cGMP accumulation was determined after coincubation with 10 μ M cGMP (0.1 μ M [ 3 H]cGMP + 9.9 μ M unlabeled cGMP) and 1–1000 μ M furosemide (a) or 10–1000 μ M bumetanide (b), respectively, for 5 min at 37°C. The furosemide and bumetanide concentrations causing half-maximal inhibitory effect (IC 50 ) on cGMP accumulation in OAT2 expressing cells were calculated. Data are presented as mean ± SEM. n furosemide = 2–4; n bumetanide = 2.
    Figure Legend Snippet: IC 50 determination for the inhibition of OAT2-mediated cGMP uptake by furosemide and bumetanide. In HEK293 cells stably transfected with OAT2 or empty vector, intracellular cGMP accumulation was determined after coincubation with 10 μ M cGMP (0.1 μ M [ 3 H]cGMP + 9.9 μ M unlabeled cGMP) and 1–1000 μ M furosemide (a) or 10–1000 μ M bumetanide (b), respectively, for 5 min at 37°C. The furosemide and bumetanide concentrations causing half-maximal inhibitory effect (IC 50 ) on cGMP accumulation in OAT2 expressing cells were calculated. Data are presented as mean ± SEM. n furosemide = 2–4; n bumetanide = 2.

    Techniques Used: Inhibition, Stable Transfection, Transfection, Plasmid Preparation, Expressing

    24) Product Images from "FKBP8 Enhances Protein Stability of the CLC-1 Chloride Channel at the Plasma Membrane"

    Article Title: FKBP8 Enhances Protein Stability of the CLC-1 Chloride Channel at the Plasma Membrane

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19123783

    Detection of cell surface FKBP8 expression by biotinylation. ( A ) Surface biotinylation experiments on HEK293T cells expressing Flag-CLC-1 in the absence or presence of Myc-FKBP8. ( Left ) Representative immunoblots. Co-expression with the Myc vector was used as the vector control (-). Cell lysates from biotinylated intact cells were subject to either direct immunoblotting analyses ( Total ) or streptavidin pull-down prior to immunoblotting ( Surface ), using the indicated antibodies (α-Myc, α-Flag, or α-GAPDH). Total represents about 8% of the amount of the protein used for streptavidin pull-down. The molecular weight markers (in kDa) are labeled to the left. GAPDH expressions are shown as the loading control. ( Right ) Quantification of total and surface CLC-1 protein levels ( n = 6). The protein density was normalized to that of the corresponding vector control; ( B ) Surface biotinylation analysis of Myc-FKBP8 in the absence or presence of Flag-CLC-1. ( Left ) Representative immunoblots. ( Right ) Quantification of FKBP8 protein levels and membrane trafficking ( n = 8). The membrane trafficking efficiency of FKBP8 was expressed as surface protein density divided by the corresponding total protein density ( Surface/total ); ( C ) Representative immunoblots showing surface biotinylation analysis of Myc-Aha1, HA-HOP, HA-Hsp90β, or Myc-Hsc70, in the absence or presence of Flag-CLC-1. Asterisks denote significant difference from the control (*, t -test: p
    Figure Legend Snippet: Detection of cell surface FKBP8 expression by biotinylation. ( A ) Surface biotinylation experiments on HEK293T cells expressing Flag-CLC-1 in the absence or presence of Myc-FKBP8. ( Left ) Representative immunoblots. Co-expression with the Myc vector was used as the vector control (-). Cell lysates from biotinylated intact cells were subject to either direct immunoblotting analyses ( Total ) or streptavidin pull-down prior to immunoblotting ( Surface ), using the indicated antibodies (α-Myc, α-Flag, or α-GAPDH). Total represents about 8% of the amount of the protein used for streptavidin pull-down. The molecular weight markers (in kDa) are labeled to the left. GAPDH expressions are shown as the loading control. ( Right ) Quantification of total and surface CLC-1 protein levels ( n = 6). The protein density was normalized to that of the corresponding vector control; ( B ) Surface biotinylation analysis of Myc-FKBP8 in the absence or presence of Flag-CLC-1. ( Left ) Representative immunoblots. ( Right ) Quantification of FKBP8 protein levels and membrane trafficking ( n = 8). The membrane trafficking efficiency of FKBP8 was expressed as surface protein density divided by the corresponding total protein density ( Surface/total ); ( C ) Representative immunoblots showing surface biotinylation analysis of Myc-Aha1, HA-HOP, HA-Hsp90β, or Myc-Hsc70, in the absence or presence of Flag-CLC-1. Asterisks denote significant difference from the control (*, t -test: p

    Techniques Used: Expressing, Western Blot, Plasmid Preparation, Molecular Weight, Labeling

    Subcellular localization of endogenous FKBP8 in HEK293T cells. ( A ) Surface biotinylation analysis of endogenous FKBP8 in the absence or presence of Flag-CLC-1 over-expression ( n = 3); ( B ) Representative biotinylation result of endogenous calnexin in the absence or presence of Flag-CLC-1 over-expression; ( C ) Representative sucrose density gradient centrifugation pattern of endogenous FKBP8 in HEK293T cells in the absence ( Flag-vector ) or presence ( Flag-CLC-1 ) of Flag-CLC-1 over-expression; ( D ) Statistical analyses of the F(3+4)/F8 ratio for the indicated membrane-associated proteins in the absence or presence of Flag-CLC-1 ( n = 4); ( E ) Representative differential centrifugation pattern of endogenous FKBP8 in HEK293T cells in the absence or presence of Flag-CLC-1 over-expression; ( F ) Statistical analyses of the 100 k/1 k ratio for the indicated membrane-associated proteins in the absence or presence of Flag-CLC-1 ( n = 4). The asterisk denotes a significant difference from the control (*, t -test: p
    Figure Legend Snippet: Subcellular localization of endogenous FKBP8 in HEK293T cells. ( A ) Surface biotinylation analysis of endogenous FKBP8 in the absence or presence of Flag-CLC-1 over-expression ( n = 3); ( B ) Representative biotinylation result of endogenous calnexin in the absence or presence of Flag-CLC-1 over-expression; ( C ) Representative sucrose density gradient centrifugation pattern of endogenous FKBP8 in HEK293T cells in the absence ( Flag-vector ) or presence ( Flag-CLC-1 ) of Flag-CLC-1 over-expression; ( D ) Statistical analyses of the F(3+4)/F8 ratio for the indicated membrane-associated proteins in the absence or presence of Flag-CLC-1 ( n = 4); ( E ) Representative differential centrifugation pattern of endogenous FKBP8 in HEK293T cells in the absence or presence of Flag-CLC-1 over-expression; ( F ) Statistical analyses of the 100 k/1 k ratio for the indicated membrane-associated proteins in the absence or presence of Flag-CLC-1 ( n = 4). The asterisk denotes a significant difference from the control (*, t -test: p

    Techniques Used: Over Expression, Gradient Centrifugation, Plasmid Preparation, Centrifugation

    FKBP8 promotes surface protein stability. ( A , B ) Representative immunoblots showing the time course of surface Flag-CLC-1 protein turn-over in the absence ( A ) or presence ( B ) of Myc-FKBP8 over-expression. Transfected HEK293T cells were subject to the indicated durations of treatment with the Golgi trafficking inhibitor brefeldin A (BFA) (5 μg/mL), followed by surface biotinylation analyses; ( C – E ) Quantification of surface Flag-CLC-1 protein stability in the absence ( black ) or presence ( red ) of Myc-FKBP8 over-expression ( n = 6–8); ( C ) Linear plot of the turn-over kinetics of surface CLC-1 protein densities. Protein density was standardized as the ratio of surface Flag-CLC-1 signal to the cognate total GAPDH signal, followed by normalization to the corresponding control at 0 hr; ( D ) Semi-logarithmic plot of the same data points, followed by linear-regression analyses ( solid lines ); ( E ) Statistical analyses of surface CLC-1 protein half-life. The surface protein turn-over time course determined from each experiment was individually plotted on a semi-logarithmic scale for linear-regression analysis, and independent protein half-life values were pooled together for statistical comparisons. The asterisk denotes a significant difference from the control (*, t -test: p
    Figure Legend Snippet: FKBP8 promotes surface protein stability. ( A , B ) Representative immunoblots showing the time course of surface Flag-CLC-1 protein turn-over in the absence ( A ) or presence ( B ) of Myc-FKBP8 over-expression. Transfected HEK293T cells were subject to the indicated durations of treatment with the Golgi trafficking inhibitor brefeldin A (BFA) (5 μg/mL), followed by surface biotinylation analyses; ( C – E ) Quantification of surface Flag-CLC-1 protein stability in the absence ( black ) or presence ( red ) of Myc-FKBP8 over-expression ( n = 6–8); ( C ) Linear plot of the turn-over kinetics of surface CLC-1 protein densities. Protein density was standardized as the ratio of surface Flag-CLC-1 signal to the cognate total GAPDH signal, followed by normalization to the corresponding control at 0 hr; ( D ) Semi-logarithmic plot of the same data points, followed by linear-regression analyses ( solid lines ); ( E ) Statistical analyses of surface CLC-1 protein half-life. The surface protein turn-over time course determined from each experiment was individually plotted on a semi-logarithmic scale for linear-regression analysis, and independent protein half-life values were pooled together for statistical comparisons. The asterisk denotes a significant difference from the control (*, t -test: p

    Techniques Used: Western Blot, Over Expression, Transfection

    25) Product Images from "TRAIL-death receptor endocytosis and apoptosis are selectively regulated by dynamin-1 activation"

    Article Title: TRAIL-death receptor endocytosis and apoptosis are selectively regulated by dynamin-1 activation

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1615072114

    Differential regulation of cargo-receptor endocytosis and TRAIL-induced apoptosis by Dyn1. ( A ) TRAIL activity (100 ng/mL) in MDA-MB-231 ( Upper ) and A549 cells ( Lower ) treated with the indicated siRNAs. ( B ) FLAG-tag TRAIL uptake or ( C ) TfnR uptake in MDA-MB-231
    Figure Legend Snippet: Differential regulation of cargo-receptor endocytosis and TRAIL-induced apoptosis by Dyn1. ( A ) TRAIL activity (100 ng/mL) in MDA-MB-231 ( Upper ) and A549 cells ( Lower ) treated with the indicated siRNAs. ( B ) FLAG-tag TRAIL uptake or ( C ) TfnR uptake in MDA-MB-231

    Techniques Used: Activity Assay, Multiple Displacement Amplification, FLAG-tag

    26) Product Images from "Programmed Cell Death Protein 5 Interacts with the Cytosolic Chaperonin Containing Tailless Complex Polypeptide 1 (CCT) to Regulate β-Tubulin Folding *Programmed Cell Death Protein 5 Interacts with the Cytosolic Chaperonin Containing Tailless Complex Polypeptide 1 (CCT) to Regulate β-Tubulin Folding * ♦"

    Article Title: Programmed Cell Death Protein 5 Interacts with the Cytosolic Chaperonin Containing Tailless Complex Polypeptide 1 (CCT) to Regulate β-Tubulin Folding *Programmed Cell Death Protein 5 Interacts with the Cytosolic Chaperonin Containing Tailless Complex Polypeptide 1 (CCT) to Regulate β-Tubulin Folding * ♦

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.542159

    PDCD5 inhibits β-tubulin folding. A , rate of association or dissociation from CCT complexes was measured by pulse-chase immunoprecipitations of CCTϵ from HEK-293T cells transfected with PDCD5-FLAG. The rate of association of CCTα and -γ subunits ( black , t ½ = 112 ± 18 min) and PDCD5 ( red , t ½ = 44 ± 2 min) was calculated along with the rate of dissociation for tubulin ( blue , t ½ = 39 ± 1 min). B , binding of β-tubulin to PDCD5 was measured by co-immunoprecipitation from HEK-293T cells transfected with FLAG-PDCD5 or empty vector. C , effect of CCT knockdown on β-tubulin binding to PDCD5 was measured by co-immunoprecipitation from HEK-293T cells treated with CCTζ siRNA or a control siRNA and later transfected with FLAG-PDCD5. The ratio of the β-tubulin band to the PDCD5 band was calculated and normalized to the control. D , folding of the indicated proteins by CCT was measured by pulse-chase co-immunoprecipitations from HEK-293T cells treated with PDCD5 siRNA or negative control as indicated (see “Experimental Procedures”). E and F , effect of PDCD5 knockdown ( E ) or overexpression ( F ) on β-tubulin binding to CCT was measured by co-immunoprecipitation with CCTϵ and immunoblotting as indicated. The ratio of the β-tubulin band to the CCTϵ band was calculated and normalized to the control. In all experiments, bars represent the average ± S.E. from at least three experiments. Representative gels or blots are shown below each graph. PDCD5 knockdown averaged between 65 and 80% as measured by immunoblotting.
    Figure Legend Snippet: PDCD5 inhibits β-tubulin folding. A , rate of association or dissociation from CCT complexes was measured by pulse-chase immunoprecipitations of CCTϵ from HEK-293T cells transfected with PDCD5-FLAG. The rate of association of CCTα and -γ subunits ( black , t ½ = 112 ± 18 min) and PDCD5 ( red , t ½ = 44 ± 2 min) was calculated along with the rate of dissociation for tubulin ( blue , t ½ = 39 ± 1 min). B , binding of β-tubulin to PDCD5 was measured by co-immunoprecipitation from HEK-293T cells transfected with FLAG-PDCD5 or empty vector. C , effect of CCT knockdown on β-tubulin binding to PDCD5 was measured by co-immunoprecipitation from HEK-293T cells treated with CCTζ siRNA or a control siRNA and later transfected with FLAG-PDCD5. The ratio of the β-tubulin band to the PDCD5 band was calculated and normalized to the control. D , folding of the indicated proteins by CCT was measured by pulse-chase co-immunoprecipitations from HEK-293T cells treated with PDCD5 siRNA or negative control as indicated (see “Experimental Procedures”). E and F , effect of PDCD5 knockdown ( E ) or overexpression ( F ) on β-tubulin binding to CCT was measured by co-immunoprecipitation with CCTϵ and immunoblotting as indicated. The ratio of the β-tubulin band to the CCTϵ band was calculated and normalized to the control. In all experiments, bars represent the average ± S.E. from at least three experiments. Representative gels or blots are shown below each graph. PDCD5 knockdown averaged between 65 and 80% as measured by immunoblotting.

    Techniques Used: Pulse Chase, Transfection, Binding Assay, Immunoprecipitation, Plasmid Preparation, Negative Control, Over Expression

    PhLP1 and PDCD5 bind CCT independently of each other. PDCD5 was either overexpressed ( A ) or knocked down ( B ), along with PhLP1-Myc overexpression in HEK-293T cells. Cell lysates were immunoprecipitated with anti-CCTϵ ( A ) or anti-Myc ( B ) and blotted as indicated. PhLP1 was either overexpressed ( C ) or knocked down ( D ), along with PDCD5-FLAG overexpression in HEK-293T cells. Cell lysates were immunoprecipitated with anti-CCTϵ ( C ) or anti-FLAG ( D ) and blotted as indicated. Bars represent the average ± S.E. from at least three experiments. Cell lysates were blotted for PDCD5-FLAG, endogenous PDCD5, PhLP1-Myc, or endogenous PhLP1 as indicated to verify the overexpression and knockdowns. Representative blots are shown below the graphs.
    Figure Legend Snippet: PhLP1 and PDCD5 bind CCT independently of each other. PDCD5 was either overexpressed ( A ) or knocked down ( B ), along with PhLP1-Myc overexpression in HEK-293T cells. Cell lysates were immunoprecipitated with anti-CCTϵ ( A ) or anti-Myc ( B ) and blotted as indicated. PhLP1 was either overexpressed ( C ) or knocked down ( D ), along with PDCD5-FLAG overexpression in HEK-293T cells. Cell lysates were immunoprecipitated with anti-CCTϵ ( C ) or anti-FLAG ( D ) and blotted as indicated. Bars represent the average ± S.E. from at least three experiments. Cell lysates were blotted for PDCD5-FLAG, endogenous PDCD5, PhLP1-Myc, or endogenous PhLP1 as indicated to verify the overexpression and knockdowns. Representative blots are shown below the graphs.

    Techniques Used: Over Expression, Immunoprecipitation

    PDCD5 forms a complex with PhLP1 and CCT. A , binding of PDCD5 to phosducin family members was measured by co-immunoprecipitation from HEK-293T cells transfected with PDCD5-FLAG along with Myc-tagged phosducin family members as indicated. After 48 h, cells were lysed, immunoprecipitated with an anti-Myc antibody, and immunoblotted for PDCD5-FLAG. B , binding of purified PDCD5 to PhLP1 or CK2-phosphorylated PhLP1 was assessed by co-immunoprecipitation in vitro . Phosphorylated PhLP1, unphosphorylated PhLP1, or no PhLP1 was incubated with PDCD5, immunoprecipitated with an Myc antibody, and blotted as indicated. C , simultaneous binding of PDCD5 and PhLP1 was measured by co-immunoprecipitation. HEK-293T cells were transfected with PDCD5-FLAG or empty vector, immunoprecipitated with FLAG, and blotted for endogenous PhLP1 and CCTϵ ( left panel ). Endogenous CCTϵ was also immunoprecipitated and blotted for endogenous PhLP1 and PDCD5-FLAG ( right panel ). A nontargeting Myc antibody served as a negative control. D , effect of CCT knockdown on PDCD5 binding to PhLP1 was measured by co-immunoprecipitation of PhLP1-Myc from HEK-293T cells treated with CCTζ siRNA or a control siRNA and later transfected with FLAG-PDCD5 and PhLP1-Myc. The ratio of the PDCD5 band to the PhLP1 band was calculated and normalized to the control. Bars represent the average ± S.E. of the mean from at least three experiments. Representative blots are shown below the graphs. E , formation of a PhLP1·PDCD5·CCT complex was demonstrated in double immunoprecipitation experiments from HEK-293T cells transfected with PDCD5-FLAG along with PhLP1-TEV-Myc or empty vector.
    Figure Legend Snippet: PDCD5 forms a complex with PhLP1 and CCT. A , binding of PDCD5 to phosducin family members was measured by co-immunoprecipitation from HEK-293T cells transfected with PDCD5-FLAG along with Myc-tagged phosducin family members as indicated. After 48 h, cells were lysed, immunoprecipitated with an anti-Myc antibody, and immunoblotted for PDCD5-FLAG. B , binding of purified PDCD5 to PhLP1 or CK2-phosphorylated PhLP1 was assessed by co-immunoprecipitation in vitro . Phosphorylated PhLP1, unphosphorylated PhLP1, or no PhLP1 was incubated with PDCD5, immunoprecipitated with an Myc antibody, and blotted as indicated. C , simultaneous binding of PDCD5 and PhLP1 was measured by co-immunoprecipitation. HEK-293T cells were transfected with PDCD5-FLAG or empty vector, immunoprecipitated with FLAG, and blotted for endogenous PhLP1 and CCTϵ ( left panel ). Endogenous CCTϵ was also immunoprecipitated and blotted for endogenous PhLP1 and PDCD5-FLAG ( right panel ). A nontargeting Myc antibody served as a negative control. D , effect of CCT knockdown on PDCD5 binding to PhLP1 was measured by co-immunoprecipitation of PhLP1-Myc from HEK-293T cells treated with CCTζ siRNA or a control siRNA and later transfected with FLAG-PDCD5 and PhLP1-Myc. The ratio of the PDCD5 band to the PhLP1 band was calculated and normalized to the control. Bars represent the average ± S.E. of the mean from at least three experiments. Representative blots are shown below the graphs. E , formation of a PhLP1·PDCD5·CCT complex was demonstrated in double immunoprecipitation experiments from HEK-293T cells transfected with PDCD5-FLAG along with PhLP1-TEV-Myc or empty vector.

    Techniques Used: Binding Assay, Immunoprecipitation, Transfection, Purification, In Vitro, Incubation, Plasmid Preparation, Negative Control

    27) Product Images from "RIPK1 protects hepatocytes from death in Fas-induced hepatitis"

    Article Title: RIPK1 protects hepatocytes from death in Fas-induced hepatitis

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-09789-8

    Fas-mediated liver injury in Ripk1 LPC-KO mice is independent of TNF-α. ( a ) Levels of serum ALT and AST (n = 4–10) in Ripk1 LPC-KO mice after 3 or 6 h mAb-Jo2 injection with a possible pre-treatment with ETA (ns: non-significant). Each circle or dot represent an individual. ( b ) Pictures of liver tissue sections, stained by H E (upper panels) or analysed by IHC for cleaved caspase-3 (lower panels), issued from Ripk1 LPC-KO mice, 6 h after mAb-Jo2 injection with a possible pre-treatment with ETA. Signal quantification of cleaved caspase-3 (lower right panel).
    Figure Legend Snippet: Fas-mediated liver injury in Ripk1 LPC-KO mice is independent of TNF-α. ( a ) Levels of serum ALT and AST (n = 4–10) in Ripk1 LPC-KO mice after 3 or 6 h mAb-Jo2 injection with a possible pre-treatment with ETA (ns: non-significant). Each circle or dot represent an individual. ( b ) Pictures of liver tissue sections, stained by H E (upper panels) or analysed by IHC for cleaved caspase-3 (lower panels), issued from Ripk1 LPC-KO mice, 6 h after mAb-Jo2 injection with a possible pre-treatment with ETA. Signal quantification of cleaved caspase-3 (lower right panel).

    Techniques Used: Mouse Assay, AST Assay, Injection, Staining, Immunohistochemistry

    RIPK1 deficiency increases the sensitivity of primary hepatocytes to Fas-agonist stimulation. Primary cultures of hepatocytes issued from Ripk1 fl/fl ( n = 2) or Ripk1 LPC-KO (n = 2) mice were subjected during 16 h to mAb-Jo2 concentrations ranging from 1 to 5 ng/mL in presence of ETA (1 µg/mL) (left panel). Additional primary cultures (n = 3 for each strains) were exposed to a unique dose of mAb-Jo2 (5 ng/mL) also in presence of ETA (1 µg/mL) for statistical analysis (middle panel). In parallel, these primary hepatocyte cultures were subjected during 16 h to mAb-Jo2 at 20 ng/mL in presence of ETA (1 µg/mL) and in absence or presence of the z-VAD-fmk pan-caspase inhibitor (right panel). Cell death was analysed by WST-1 based assay and data are expressed as a percentage of signal obtained in basal survival conditions without Fas-agonist. Error bars corresponds either to internal triplicates for each primary cultures (left panel) or to triplicates of independent primary cultures (middle and right panels).
    Figure Legend Snippet: RIPK1 deficiency increases the sensitivity of primary hepatocytes to Fas-agonist stimulation. Primary cultures of hepatocytes issued from Ripk1 fl/fl ( n = 2) or Ripk1 LPC-KO (n = 2) mice were subjected during 16 h to mAb-Jo2 concentrations ranging from 1 to 5 ng/mL in presence of ETA (1 µg/mL) (left panel). Additional primary cultures (n = 3 for each strains) were exposed to a unique dose of mAb-Jo2 (5 ng/mL) also in presence of ETA (1 µg/mL) for statistical analysis (middle panel). In parallel, these primary hepatocyte cultures were subjected during 16 h to mAb-Jo2 at 20 ng/mL in presence of ETA (1 µg/mL) and in absence or presence of the z-VAD-fmk pan-caspase inhibitor (right panel). Cell death was analysed by WST-1 based assay and data are expressed as a percentage of signal obtained in basal survival conditions without Fas-agonist. Error bars corresponds either to internal triplicates for each primary cultures (left panel) or to triplicates of independent primary cultures (middle and right panels).

    Techniques Used: Mouse Assay, WST-1 Assay

    RIPK1 deficiency sensitizes mice to Fas-mediated liver injuries. ( a ) Levels of serum ALT and AST, 3 and 6 h after mAb-Jo2 injection in Ripk1 fl/fl and Ripk1 LPC-KO mice (n = 6–7). ( b ) Pictures of liver tissue sections, stained by H E (upper panels) or analysed by TUNEL (in red) and DAPI (in blue) immunofluorescence (lower panels) issued from Ripk1 fl/fl and Ripk1 LPC-KO mice, 6 h after mAb-Jo2 injection. Yellow arrows show necrotic areas, PV: portal vein. ( c ) Immunostaining of cleaved caspase-3 in the livers of Ripk1 fl/fl and Ripk1 LPC-KO mice, 6 h after mAb-Jo2 injection. ( d ) Mean levels of cleaved caspase-3 (left panel) and of JNK phosphorylation status (right panel) in the livers of Ripk1 fl/fl (n = 5) and Ripk1 LPC-KO (n = 5) mice, collected 6 h after mAb-Jo2 injection (see corresponding Western blots in Supplementary Fig. S1 ). ( e ) Levels of hepatic IL-1β, IL-6 and TNF-α transcripts in Ripk1 fl/fl or Ripk1 LPC-KO mice, 6 h after PBS (n = 3 mice) or mAb-Jo2 injection (n = 6–7 mice). For all graphs, each circle represents an individual.
    Figure Legend Snippet: RIPK1 deficiency sensitizes mice to Fas-mediated liver injuries. ( a ) Levels of serum ALT and AST, 3 and 6 h after mAb-Jo2 injection in Ripk1 fl/fl and Ripk1 LPC-KO mice (n = 6–7). ( b ) Pictures of liver tissue sections, stained by H E (upper panels) or analysed by TUNEL (in red) and DAPI (in blue) immunofluorescence (lower panels) issued from Ripk1 fl/fl and Ripk1 LPC-KO mice, 6 h after mAb-Jo2 injection. Yellow arrows show necrotic areas, PV: portal vein. ( c ) Immunostaining of cleaved caspase-3 in the livers of Ripk1 fl/fl and Ripk1 LPC-KO mice, 6 h after mAb-Jo2 injection. ( d ) Mean levels of cleaved caspase-3 (left panel) and of JNK phosphorylation status (right panel) in the livers of Ripk1 fl/fl (n = 5) and Ripk1 LPC-KO (n = 5) mice, collected 6 h after mAb-Jo2 injection (see corresponding Western blots in Supplementary Fig. S1 ). ( e ) Levels of hepatic IL-1β, IL-6 and TNF-α transcripts in Ripk1 fl/fl or Ripk1 LPC-KO mice, 6 h after PBS (n = 3 mice) or mAb-Jo2 injection (n = 6–7 mice). For all graphs, each circle represents an individual.

    Techniques Used: Mouse Assay, AST Assay, Injection, Staining, TUNEL Assay, Immunofluorescence, Immunostaining, Western Blot

    28) Product Images from "A highly sensitive FRET-based approach reveals secretion of the actin-binding protein toxofilin during Toxoplasma gondii infection"

    Article Title: A highly sensitive FRET-based approach reveals secretion of the actin-binding protein toxofilin during Toxoplasma gondii infection

    Journal: Cellular microbiology

    doi: 10.1111/j.1462-5822.2009.01378.x

    Δ txf parasites have similar invasion efficiency and in vitro growth as control parasites. (A) The invasion of Δ txf or control parasites in HFF was synchronized by treatment with high potassium buffer and a shift to DMEM containing lower potassium for the indicated time periods to allow parasite invasion. As the Δ txf and control parasites are GFP + , the invasion efficiency was scored by counting the number of intracellular parasites (GFP + SAG1 − ) relative to the total number of parasites in each field. This experiment was performed six times with comparable results; a representative experiment is shown. The error bars represent the standard deviation of intracellular parasites in 10 independent fields. (B) HFF were infected with Δ txf or control parasites for 2 hours and washed to remove uninvaded parasites. 24 hours later the cells were fixed and the number of parasites per vacuole in 8 fields was counted and expressed as a percentage of the total number of parasites in the field. The error bars represent the standard deviation from four biological replicate wells. This experiment was performed three times as a time course, and the data shown are a representative result. (C) HFF were infected with control or Δ txf parasites, and at 30 hours post-infection, the calcium ionophore A23187 was added to the cells for 15, 30, 60 or 90 seconds. For each time point, the number of lysed vacuoles was counted in 5 fields and expressed as a percentage of the total number of vacuoles in the field. This experiment was performed 5 times on different days, and all of the data are shown. An adjusted Wald F-test was used to calculate the p-values (p = 0.3 at 15 seconds, p = 0.74 at 30 seconds, p = 0.41 at 60 seconds, p = 0.54 at 90 seconds).
    Figure Legend Snippet: Δ txf parasites have similar invasion efficiency and in vitro growth as control parasites. (A) The invasion of Δ txf or control parasites in HFF was synchronized by treatment with high potassium buffer and a shift to DMEM containing lower potassium for the indicated time periods to allow parasite invasion. As the Δ txf and control parasites are GFP + , the invasion efficiency was scored by counting the number of intracellular parasites (GFP + SAG1 − ) relative to the total number of parasites in each field. This experiment was performed six times with comparable results; a representative experiment is shown. The error bars represent the standard deviation of intracellular parasites in 10 independent fields. (B) HFF were infected with Δ txf or control parasites for 2 hours and washed to remove uninvaded parasites. 24 hours later the cells were fixed and the number of parasites per vacuole in 8 fields was counted and expressed as a percentage of the total number of parasites in the field. The error bars represent the standard deviation from four biological replicate wells. This experiment was performed three times as a time course, and the data shown are a representative result. (C) HFF were infected with control or Δ txf parasites, and at 30 hours post-infection, the calcium ionophore A23187 was added to the cells for 15, 30, 60 or 90 seconds. For each time point, the number of lysed vacuoles was counted in 5 fields and expressed as a percentage of the total number of vacuoles in the field. This experiment was performed 5 times on different days, and all of the data are shown. An adjusted Wald F-test was used to calculate the p-values (p = 0.3 at 15 seconds, p = 0.74 at 30 seconds, p = 0.41 at 60 seconds, p = 0.54 at 90 seconds).

    Techniques Used: In Vitro, Standard Deviation, Infection

    29) Product Images from "Hydrogen Sulfide Improves Cardiomyocyte Function in a Cardiac Arrest Model"

    Article Title: Hydrogen Sulfide Improves Cardiomyocyte Function in a Cardiac Arrest Model

    Journal: Annals of Transplantation

    doi: 10.12659/AOT.901410

    Effect of GYY4137 on ATP content and protein synthesis in HL-1 cells. ( A ) Relative ATP content during preconditioning. ATP content is represented as relative content to non-preconditioned cells (0 hour). Dashed line represents cells treated with active GYY4137 and solid line represents cells treated with the inactive GYY4137. ( B ) Top panel , dot blot assay showing methionine L-azidohomoalanine (AHA) incorporation at 3 hours of preconditioning. Bottom panel , experimental set up for AHA incorporation: Negative control, non-biotinylated AHA; positive control, exponential cell growth in complete Claycomb medium; no analogue, biotinylation of extract without AHA incorporation; actidione, exponential cell growth in complete Claycomb medium after pre-treatment with the protein synthesis inhibitor cycloheximide. ( C ) Relative ATP content during nutrient starvation. ATP content is represented as relative content to the initial level of control cells (0 hour). Dashed line represents cells treated with active GYY4137 and solid line represents cells treated with inactive GYY4137. ( D ) Dot blot assay showing AHA incorporation at 30 minutes (0.5 hours), 1 hour, and 3 hours after starvation. * p
    Figure Legend Snippet: Effect of GYY4137 on ATP content and protein synthesis in HL-1 cells. ( A ) Relative ATP content during preconditioning. ATP content is represented as relative content to non-preconditioned cells (0 hour). Dashed line represents cells treated with active GYY4137 and solid line represents cells treated with the inactive GYY4137. ( B ) Top panel , dot blot assay showing methionine L-azidohomoalanine (AHA) incorporation at 3 hours of preconditioning. Bottom panel , experimental set up for AHA incorporation: Negative control, non-biotinylated AHA; positive control, exponential cell growth in complete Claycomb medium; no analogue, biotinylation of extract without AHA incorporation; actidione, exponential cell growth in complete Claycomb medium after pre-treatment with the protein synthesis inhibitor cycloheximide. ( C ) Relative ATP content during nutrient starvation. ATP content is represented as relative content to the initial level of control cells (0 hour). Dashed line represents cells treated with active GYY4137 and solid line represents cells treated with inactive GYY4137. ( D ) Dot blot assay showing AHA incorporation at 30 minutes (0.5 hours), 1 hour, and 3 hours after starvation. * p

    Techniques Used: Dot Blot, Negative Control, Positive Control

    GYY4137 improves cardioplegic cardio-protective performance during cardiac arrest. ( A ) Scheme of the procedure to test cardioplegic solutions in rat hearts utilizing a Langendorff apparatus. ( B ) Western blotting for caspase-3 in rat heart tissues perfused with Conv or del Nido cardioplegic solutions with or without GYY4137 (n=4). The graph shows quantification of densitometry analysis utilizing ImageJ software. ( C ) ATP quantification in rat heart tissue after finalization of Langendorff perfusion protocol as described in methods section. For the sham group, rat hearts were not subjected to the experimental Langendorff protocol; the organs were directly explanted and ATP content was analyzed. * p
    Figure Legend Snippet: GYY4137 improves cardioplegic cardio-protective performance during cardiac arrest. ( A ) Scheme of the procedure to test cardioplegic solutions in rat hearts utilizing a Langendorff apparatus. ( B ) Western blotting for caspase-3 in rat heart tissues perfused with Conv or del Nido cardioplegic solutions with or without GYY4137 (n=4). The graph shows quantification of densitometry analysis utilizing ImageJ software. ( C ) ATP quantification in rat heart tissue after finalization of Langendorff perfusion protocol as described in methods section. For the sham group, rat hearts were not subjected to the experimental Langendorff protocol; the organs were directly explanted and ATP content was analyzed. * p

    Techniques Used: Western Blot, Software

    Effect of GYY4137 on HL-1 cell apoptosis. ( A ) Effect of GYY4137 preconditioning on apoptosis of cells cultured in glucose and amino acid deprivation medium (-Glu KH). ( B ) Effect of GYY4137 preconditioning on apoptosis of cells cultured in amino acid deprivation medium (KH). ( C, D ) Effect of GYY4137 preconditioning on apoptosis of cells treated with staurosporine ( C ) and doxorubicin ( D ). ( E ) Effect of GYY4137 on apoptosis of cells during starvation in -Glu KH medium without preconditioning. In all cases, results show percentage of apoptosis over time. Dashed lines represent cells treated with active GYY4137 and solid lines represent cells treated with inactive GYY4137. Three replicates in three differences experiments were performed per condition. * p
    Figure Legend Snippet: Effect of GYY4137 on HL-1 cell apoptosis. ( A ) Effect of GYY4137 preconditioning on apoptosis of cells cultured in glucose and amino acid deprivation medium (-Glu KH). ( B ) Effect of GYY4137 preconditioning on apoptosis of cells cultured in amino acid deprivation medium (KH). ( C, D ) Effect of GYY4137 preconditioning on apoptosis of cells treated with staurosporine ( C ) and doxorubicin ( D ). ( E ) Effect of GYY4137 on apoptosis of cells during starvation in -Glu KH medium without preconditioning. In all cases, results show percentage of apoptosis over time. Dashed lines represent cells treated with active GYY4137 and solid lines represent cells treated with inactive GYY4137. Three replicates in three differences experiments were performed per condition. * p

    Techniques Used: Cell Culture

    Short-term electrical recovery after GYY4137 treatment in the isolated Langendorff-perfused rat model. ( A ) Representative 5 second volume-conducted pseudo-ECG traces showing pre-treatment (Pre, light) or post-treatment (Post, dark) with cardioplegic solutions. A better preservation of heart rate (HR) and more regular cardiac activation is detected with del Nido+GYY4137 formulation than with untreated KH solution-preserved hearts. Traces are segmented after 15 minutes of registration from a single-lead electrode located at the epicardial base of the right ventricle in the Langendorff-perfused whole-heart. ( B ) Quantification of the effects of cardioplegic treatment on heart rate (HR, beats per minute) reveals significant slowing of cardiac activation, whereas heartrate-slowing is non-significantly different in the del Nido + GYY4137 treated group. * p
    Figure Legend Snippet: Short-term electrical recovery after GYY4137 treatment in the isolated Langendorff-perfused rat model. ( A ) Representative 5 second volume-conducted pseudo-ECG traces showing pre-treatment (Pre, light) or post-treatment (Post, dark) with cardioplegic solutions. A better preservation of heart rate (HR) and more regular cardiac activation is detected with del Nido+GYY4137 formulation than with untreated KH solution-preserved hearts. Traces are segmented after 15 minutes of registration from a single-lead electrode located at the epicardial base of the right ventricle in the Langendorff-perfused whole-heart. ( B ) Quantification of the effects of cardioplegic treatment on heart rate (HR, beats per minute) reveals significant slowing of cardiac activation, whereas heartrate-slowing is non-significantly different in the del Nido + GYY4137 treated group. * p

    Techniques Used: Isolation, Preserving, Activation Assay

    GYY4137 reduces oxidative stress during cardiac arrest. ( A ) Levels of oxidative stress biomarkers GSH, GSSG; and ( B ) SAM and SAH were analyzed in rat hearts treated with Conv or del Nido cardioplegia solutions with or without GYY4137. Graphs show the ratio of GSH/GSSG ( A ) and SAM/SAH ( B ). Four-six experiments were performed per condition. * p
    Figure Legend Snippet: GYY4137 reduces oxidative stress during cardiac arrest. ( A ) Levels of oxidative stress biomarkers GSH, GSSG; and ( B ) SAM and SAH were analyzed in rat hearts treated with Conv or del Nido cardioplegia solutions with or without GYY4137. Graphs show the ratio of GSH/GSSG ( A ) and SAM/SAH ( B ). Four-six experiments were performed per condition. * p

    Techniques Used:

    30) Product Images from "Novel Insights into the Molecular Mechanism of Action of DNA Hypomethylating Agents"

    Article Title: Novel Insights into the Molecular Mechanism of Action of DNA Hypomethylating Agents

    Journal: Genes & Cancer

    doi: 10.1177/1947601912452665

    N-terminal 118 amino acids is necessary but not sufficient for decitabine-induced degradation of Dnmt1. H293T and HCT116 cells were transiently transfected with a Flag-tagged N-terminal 118–amino acid sequence of the Dnmt1 protein. After 24 hours,
    Figure Legend Snippet: N-terminal 118 amino acids is necessary but not sufficient for decitabine-induced degradation of Dnmt1. H293T and HCT116 cells were transiently transfected with a Flag-tagged N-terminal 118–amino acid sequence of the Dnmt1 protein. After 24 hours,

    Techniques Used: Transfection, Sequencing

    Decitabine treatment facilitates DNMT1 phosphorylation that can be blocked by PKCδ inhibition. ( A ) DNMT1 protein is phosphorylated at serine moiety in vivo . HCT116 cells were pretreated with the proteasomal inhibitor MG-132 for 15 minutes followed
    Figure Legend Snippet: Decitabine treatment facilitates DNMT1 phosphorylation that can be blocked by PKCδ inhibition. ( A ) DNMT1 protein is phosphorylated at serine moiety in vivo . HCT116 cells were pretreated with the proteasomal inhibitor MG-132 for 15 minutes followed

    Techniques Used: Inhibition, In Vivo

    Decitabine-induced degradation of DNMT1 is blocked by protein kinase C inhibitors. ( A ) HCT116 cells were pretreated with inhibitors of the MAP kinase pathway (PD98059, U0126, SP600125, SB203580), tyrosine kinase (genistein), PI3K (LY294002), a broad-spectrum
    Figure Legend Snippet: Decitabine-induced degradation of DNMT1 is blocked by protein kinase C inhibitors. ( A ) HCT116 cells were pretreated with inhibitors of the MAP kinase pathway (PD98059, U0126, SP600125, SB203580), tyrosine kinase (genistein), PI3K (LY294002), a broad-spectrum

    Techniques Used:

    DNMT1 is predominantly associated with oligonucleosomes in HCT116 cells, and decitabine treatment causes depletion but does not shift it to polynucleosomes. ( A, B ) HCT116 cells were treated with decitabine (1 µM) for 12 hours. Nuclei isolated
    Figure Legend Snippet: DNMT1 is predominantly associated with oligonucleosomes in HCT116 cells, and decitabine treatment causes depletion but does not shift it to polynucleosomes. ( A, B ) HCT116 cells were treated with decitabine (1 µM) for 12 hours. Nuclei isolated

    Techniques Used: Isolation

    31) Product Images from "Basolateral membrane targeting of a renal-epithelial inwardly rectifying potassium channel from the cortical collecting duct, CCD-IRK3, in MDCK cells"

    Article Title: Basolateral membrane targeting of a renal-epithelial inwardly rectifying potassium channel from the cortical collecting duct, CCD-IRK3, in MDCK cells

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi:

    Biochemical analysis of epitope-tagged IRK3 channels. Immunoprecipitation of radiolabeled tagged IRK3 channel proteins with P5D4 ( A ) and AU1 ( B ) antibodies. A specific 57-kDa band was immunoprecipitated in VSV, VSV(Y2F), and AU1-IRK3-MDCK cells ( A , lanes 2 and 3, and B , lane 2, respectively) but not in the absence of P5D4 and AU1 antibodies ( A , lanes 4 and 5, and B , lane 3, respectively) nor in MDCK cells ( A , lane 1, and B , lane 1). ( C ) VSV-IRK3 protein (lane 1) was not sensitive to PNGaseF and endo H treatment (lanes 2 and 3, respectively). Numbers on the left indicate mobility of prestained molecular mass standards in kDa.
    Figure Legend Snippet: Biochemical analysis of epitope-tagged IRK3 channels. Immunoprecipitation of radiolabeled tagged IRK3 channel proteins with P5D4 ( A ) and AU1 ( B ) antibodies. A specific 57-kDa band was immunoprecipitated in VSV, VSV(Y2F), and AU1-IRK3-MDCK cells ( A , lanes 2 and 3, and B , lane 2, respectively) but not in the absence of P5D4 and AU1 antibodies ( A , lanes 4 and 5, and B , lane 3, respectively) nor in MDCK cells ( A , lane 1, and B , lane 1). ( C ) VSV-IRK3 protein (lane 1) was not sensitive to PNGaseF and endo H treatment (lanes 2 and 3, respectively). Numbers on the left indicate mobility of prestained molecular mass standards in kDa.

    Techniques Used: Immunoprecipitation

    32) Product Images from "Hydrogen Sulfide Improves Cardiomyocyte Function in a Cardiac Arrest Model"

    Article Title: Hydrogen Sulfide Improves Cardiomyocyte Function in a Cardiac Arrest Model

    Journal: Annals of Transplantation

    doi: 10.12659/AOT.901410

    Effect of GYY4137 on ATP content and protein synthesis in HL-1 cells. ( A ) Relative ATP content during preconditioning. ATP content is represented as relative content to non-preconditioned cells (0 hour). Dashed line represents cells treated with active GYY4137 and solid line represents cells treated with the inactive GYY4137. ( B ) Top panel , dot blot assay showing methionine L-azidohomoalanine (AHA) incorporation at 3 hours of preconditioning. Bottom panel , experimental set up for AHA incorporation: Negative control, non-biotinylated AHA; positive control, exponential cell growth in complete Claycomb medium; no analogue, biotinylation of extract without AHA incorporation; actidione, exponential cell growth in complete Claycomb medium after pre-treatment with the protein synthesis inhibitor cycloheximide. ( C ) Relative ATP content during nutrient starvation. ATP content is represented as relative content to the initial level of control cells (0 hour). Dashed line represents cells treated with active GYY4137 and solid line represents cells treated with inactive GYY4137. ( D ) Dot blot assay showing AHA incorporation at 30 minutes (0.5 hours), 1 hour, and 3 hours after starvation. * p
    Figure Legend Snippet: Effect of GYY4137 on ATP content and protein synthesis in HL-1 cells. ( A ) Relative ATP content during preconditioning. ATP content is represented as relative content to non-preconditioned cells (0 hour). Dashed line represents cells treated with active GYY4137 and solid line represents cells treated with the inactive GYY4137. ( B ) Top panel , dot blot assay showing methionine L-azidohomoalanine (AHA) incorporation at 3 hours of preconditioning. Bottom panel , experimental set up for AHA incorporation: Negative control, non-biotinylated AHA; positive control, exponential cell growth in complete Claycomb medium; no analogue, biotinylation of extract without AHA incorporation; actidione, exponential cell growth in complete Claycomb medium after pre-treatment with the protein synthesis inhibitor cycloheximide. ( C ) Relative ATP content during nutrient starvation. ATP content is represented as relative content to the initial level of control cells (0 hour). Dashed line represents cells treated with active GYY4137 and solid line represents cells treated with inactive GYY4137. ( D ) Dot blot assay showing AHA incorporation at 30 minutes (0.5 hours), 1 hour, and 3 hours after starvation. * p

    Techniques Used: Dot Blot, Negative Control, Positive Control

    33) Product Images from "Protein Traffic Disorders: an Effective High-Throughput Fluorescence Microscopy Pipeline for Drug Discovery"

    Article Title: Protein Traffic Disorders: an Effective High-Throughput Fluorescence Microscopy Pipeline for Drug Discovery

    Journal: Scientific Reports

    doi: 10.1038/srep09038

    Microscopy features of the CFBE cell lines expressing mCherry-Flag-wt-CFTR (a–d) or mCherry-Flag-F508del-CFTR (e–h). Cells were grown in the presence of 1 μg/ml Dox so as to induce CFTR expression. DIC bright field images (a, e) showing an overall cell morphology reminiscent of CFTR localization, especially for F508del-CFTR, where its ER accumulation is visible. Widefield fluorescence images show triply labelled unpermeabilized cells. Nuclei are stained with hoechst 33342 (b, f), mCherry fluorescence is proportional to the total amount of expressed CFTR (c, g) and Alexa Fluor® 647 (immuno)fluorescence is proportional to the amount of Flag tags exposed extracellularly ( i.e. CFTR molecules present at the PM) (d, h). Fluorescence images were obtained under equivalent conditions for both cell lines. Scale bar = 20 μm.
    Figure Legend Snippet: Microscopy features of the CFBE cell lines expressing mCherry-Flag-wt-CFTR (a–d) or mCherry-Flag-F508del-CFTR (e–h). Cells were grown in the presence of 1 μg/ml Dox so as to induce CFTR expression. DIC bright field images (a, e) showing an overall cell morphology reminiscent of CFTR localization, especially for F508del-CFTR, where its ER accumulation is visible. Widefield fluorescence images show triply labelled unpermeabilized cells. Nuclei are stained with hoechst 33342 (b, f), mCherry fluorescence is proportional to the total amount of expressed CFTR (c, g) and Alexa Fluor® 647 (immuno)fluorescence is proportional to the amount of Flag tags exposed extracellularly ( i.e. CFTR molecules present at the PM) (d, h). Fluorescence images were obtained under equivalent conditions for both cell lines. Scale bar = 20 μm.

    Techniques Used: Microscopy, Expressing, Fluorescence, Staining

    34) Product Images from "Differential sorting behavior for soluble and transmembrane cargoes at the trans-Golgi network in endocrine cells"

    Article Title: Differential sorting behavior for soluble and transmembrane cargoes at the trans-Golgi network in endocrine cells

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E19-10-0561

    INS-1 cells were transfected as in Figure 1 with the indicated cargoes. Fluorescence within the TGN region (A) and the ER (B) was monitored and the data were fitted using a first-order kinetic model to extrapolate ER (k ER ) and TGN rate constants (C). Representative curves are shown for each cargo ( n = 16 for NPY-emdGFP-SBP from three independent transfections; n = 8 for SgII-emdGFP-SBP from two independent transfections; n = 14 for phogrin-emdGFP-SBP from three independent transfections; n = 11 for VMAT2-emdGFP-SBP from two independent transfections).
    Figure Legend Snippet: INS-1 cells were transfected as in Figure 1 with the indicated cargoes. Fluorescence within the TGN region (A) and the ER (B) was monitored and the data were fitted using a first-order kinetic model to extrapolate ER (k ER ) and TGN rate constants (C). Representative curves are shown for each cargo ( n = 16 for NPY-emdGFP-SBP from three independent transfections; n = 8 for SgII-emdGFP-SBP from two independent transfections; n = 14 for phogrin-emdGFP-SBP from three independent transfections; n = 11 for VMAT2-emdGFP-SBP from two independent transfections).

    Techniques Used: Transfection, Fluorescence

    (A) INS-1 cells were transfected with NPY-emdGFP-SBP together with the TGN marker sialyltransferase-TagRFP657 and ER-hook, incubated with biotin for the indicated times, fixed, and imaged using a widefield epifluorescence microscope. (B) INS-1 cells transfected with RUSH constructs were incubated with biotin for 1 or 24 h, fixed, and immunostained for insulin followed by Alexa Fluor 647–conjugated secondary antibodies. Cells were imaged by spinning-disk confocal microscopy. Insets denoted by a square are shown in B’. INS-1 (C, D) or PC12 cells (E) were transfected with NPY-emdGFP-SBP and ER hook for secretion assays. Cells were incubated with biotin for 1.5 h (C, E) or 3 h (D) followed by exposure to basal or stimulated conditions for 15 min. Secretion was determined by reading the fluorescence in the media and was normalized to total fluorescence in cell lysate. * p
    Figure Legend Snippet: (A) INS-1 cells were transfected with NPY-emdGFP-SBP together with the TGN marker sialyltransferase-TagRFP657 and ER-hook, incubated with biotin for the indicated times, fixed, and imaged using a widefield epifluorescence microscope. (B) INS-1 cells transfected with RUSH constructs were incubated with biotin for 1 or 24 h, fixed, and immunostained for insulin followed by Alexa Fluor 647–conjugated secondary antibodies. Cells were imaged by spinning-disk confocal microscopy. Insets denoted by a square are shown in B’. INS-1 (C, D) or PC12 cells (E) were transfected with NPY-emdGFP-SBP and ER hook for secretion assays. Cells were incubated with biotin for 1.5 h (C, E) or 3 h (D) followed by exposure to basal or stimulated conditions for 15 min. Secretion was determined by reading the fluorescence in the media and was normalized to total fluorescence in cell lysate. * p

    Techniques Used: Transfection, Marker, Incubation, Microscopy, Construct, Confocal Microscopy, Fluorescence

    (A–C) INS-1 cells cotransfected with NPY-mCherry-SBP and indicated emdGFP-RUSH constructs as in Figure 2 and imaged every 300 ms for 2 min after 40 min of biotin treatment. The proportion of vesicles positive for both markers was determined (A, B); * p
    Figure Legend Snippet: (A–C) INS-1 cells cotransfected with NPY-mCherry-SBP and indicated emdGFP-RUSH constructs as in Figure 2 and imaged every 300 ms for 2 min after 40 min of biotin treatment. The proportion of vesicles positive for both markers was determined (A, B); * p

    Techniques Used: Construct

    INS-1 cells transfected with the indicated cargoes were incubated with biotin in presence or absence of 80 μM dynasore. Fluorescence within the TGN region was monitored as described in Figure 2A . Insets are shown in A’, C’, and E’. Arrows indicate post-TGN carriers. Average curves of TGN fluorescence are shown in B, D, and F ( n = 9 and 9 cells from two independent transfections for SBP-emdGFP-VSV-G vehicle and dynasore, respectively; n = 19 and 20 cells from independent transfections for NPY-emdGFP-SBP vehicle and dynasore, respectively; n = 7 and 12 cells from three independent transfections for phogrin-emdGFP-SBP vehicle and dynasore, respectively). Data shown indicate mean ± SEM. Scale bar indicates 10 μm and 1 μm for insets.
    Figure Legend Snippet: INS-1 cells transfected with the indicated cargoes were incubated with biotin in presence or absence of 80 μM dynasore. Fluorescence within the TGN region was monitored as described in Figure 2A . Insets are shown in A’, C’, and E’. Arrows indicate post-TGN carriers. Average curves of TGN fluorescence are shown in B, D, and F ( n = 9 and 9 cells from two independent transfections for SBP-emdGFP-VSV-G vehicle and dynasore, respectively; n = 19 and 20 cells from independent transfections for NPY-emdGFP-SBP vehicle and dynasore, respectively; n = 7 and 12 cells from three independent transfections for phogrin-emdGFP-SBP vehicle and dynasore, respectively). Data shown indicate mean ± SEM. Scale bar indicates 10 μm and 1 μm for insets.

    Techniques Used: Transfection, Incubation, Fluorescence

    INS-1 cells were transfected with phogrin-emdGFP-SBP (A) or SBP-emdGFP-VSV-G (B) and incubated with biotin as in Figure 1 . (A) INS-1 cells were costained for insulin at 1 or 24 h. Insets denoted by a square are shown in A’ and C’. The extent of colocalization with insulin was determined by MCC at 1 h (B) and 24 h (D). MCC was also calculated for NPY-emdGFP-SBP for comparison using the images shown in Figure 1 ; * p
    Figure Legend Snippet: INS-1 cells were transfected with phogrin-emdGFP-SBP (A) or SBP-emdGFP-VSV-G (B) and incubated with biotin as in Figure 1 . (A) INS-1 cells were costained for insulin at 1 or 24 h. Insets denoted by a square are shown in A’ and C’. The extent of colocalization with insulin was determined by MCC at 1 h (B) and 24 h (D). MCC was also calculated for NPY-emdGFP-SBP for comparison using the images shown in Figure 1 ; * p

    Techniques Used: Transfection, Incubation

    35) Product Images from "Factor VII Light Chain-Targeted Lidamycin Shows Intensified Therapeutic Efficacy for Liver Cancer"

    Article Title: Factor VII Light Chain-Targeted Lidamycin Shows Intensified Therapeutic Efficacy for Liver Cancer

    Journal: Cancer Biotherapy & Radiopharmaceuticals

    doi: 10.1089/cbr.2012.1209

    Chromatin condensation and cleavage of genomic DNA in human liver cancer HepG2 cells induced by hlFVII-LDP-AE were determined by staining with the fluorescent dye Hoechst 33342 and TUNEL assay, respectively. (A) Chromatin condensation assay. (B) TUNEL
    Figure Legend Snippet: Chromatin condensation and cleavage of genomic DNA in human liver cancer HepG2 cells induced by hlFVII-LDP-AE were determined by staining with the fluorescent dye Hoechst 33342 and TUNEL assay, respectively. (A) Chromatin condensation assay. (B) TUNEL

    Techniques Used: Staining, TUNEL Assay

    36) Product Images from "ZEB1‐mediated melanoma cell plasticity enhances resistance to MAPK inhibitors"

    Article Title: ZEB1‐mediated melanoma cell plasticity enhances resistance to MAPK inhibitors

    Journal: EMBO Molecular Medicine

    doi: 10.15252/emmm.201505971

    High levels of ZEB 1 expression are correlated with low MITF levels and are associated with inherent resistance to MAPK i in BRAF V 600 ‐mutated melanoma cell lines MITF mRNA expression according to ZEB1 expression levels in 61 melanoma cell lines available through the CCLE (Pearson correlation test). ZEB1, ZEB2, TWIST1, and MITF expression in a panel of BRAF V600 ‐mutated melanoma cells assessed by Western blot. GLO and C‐09.10 cells are patient‐derived short‐term cultures. Actin was used as a loading control. Quantitative PCR analyses of ZEB1 , ZEB2 , TWIST1, and MITF in the same panel of cell lines. mRNA expression levels are represented relative to C‐09.10 cells, in which the levels were arbitrarily fixed at 1 ( n = 3, mean ± SD). The dotted line separates ZEB1 high (left) and ZEB1 low (right) cell lines. ZEB1 , ZEB2 , TWIST1 , and MITF mRNA expression according to the IC 50 of the drug (μM) administered (BRAFi/MEKi), in melanoma cell lines from the CCLE ( n = 28) (Tukey box plot, Student's t ‐test). High ZEB1 , low ZEB2 , and low MITF expression levels were correlated with BRAFi (PLX4720) and MEKi (AZD6244) resistance. PLX4720 is an analog of PLX4032. IC 50 values of PLX4032 (μM) in the panel of BRAF V600 melanoma cells as determined by ATP assay ( n = 3, mean ± SD). For SKMEL24 and WM793, IC 50 was > 8 μM. Source data are available online for this figure.
    Figure Legend Snippet: High levels of ZEB 1 expression are correlated with low MITF levels and are associated with inherent resistance to MAPK i in BRAF V 600 ‐mutated melanoma cell lines MITF mRNA expression according to ZEB1 expression levels in 61 melanoma cell lines available through the CCLE (Pearson correlation test). ZEB1, ZEB2, TWIST1, and MITF expression in a panel of BRAF V600 ‐mutated melanoma cells assessed by Western blot. GLO and C‐09.10 cells are patient‐derived short‐term cultures. Actin was used as a loading control. Quantitative PCR analyses of ZEB1 , ZEB2 , TWIST1, and MITF in the same panel of cell lines. mRNA expression levels are represented relative to C‐09.10 cells, in which the levels were arbitrarily fixed at 1 ( n = 3, mean ± SD). The dotted line separates ZEB1 high (left) and ZEB1 low (right) cell lines. ZEB1 , ZEB2 , TWIST1 , and MITF mRNA expression according to the IC 50 of the drug (μM) administered (BRAFi/MEKi), in melanoma cell lines from the CCLE ( n = 28) (Tukey box plot, Student's t ‐test). High ZEB1 , low ZEB2 , and low MITF expression levels were correlated with BRAFi (PLX4720) and MEKi (AZD6244) resistance. PLX4720 is an analog of PLX4032. IC 50 values of PLX4032 (μM) in the panel of BRAF V600 melanoma cells as determined by ATP assay ( n = 3, mean ± SD). For SKMEL24 and WM793, IC 50 was > 8 μM. Source data are available online for this figure.

    Techniques Used: Expressing, Western Blot, Derivative Assay, Real-time Polymerase Chain Reaction, ATP Assay

    37) Product Images from "Bovine Organospecific Microvascular Endothelial Cell Lines as New and Relevant In Vitro Models to Study Viral Infections"

    Article Title: Bovine Organospecific Microvascular Endothelial Cell Lines as New and Relevant In Vitro Models to Study Viral Infections

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms21155249

    Main endothelial cell markers expressed in various bovine cell lines assessed by flow cytometry. Bovine endothelial cell lines and two human endothelial cell lines, as controls, were labeled with anti-CD143, anti-CD146, and anti-CXCR4 antibodies and then analyzed by flow cytometry. Results are shown as histograms showing the fluorescence intensity with the isotypic control in dark blue and the antibody labeling in green.
    Figure Legend Snippet: Main endothelial cell markers expressed in various bovine cell lines assessed by flow cytometry. Bovine endothelial cell lines and two human endothelial cell lines, as controls, were labeled with anti-CD143, anti-CD146, and anti-CXCR4 antibodies and then analyzed by flow cytometry. Results are shown as histograms showing the fluorescence intensity with the isotypic control in dark blue and the antibody labeling in green.

    Techniques Used: Flow Cytometry, Labeling, Fluorescence, Antibody Labeling

    38) Product Images from "Activated Abl kinase inhibits oncogenic transforming growth factor-? signaling and tumorigenesis in mammary tumors"

    Article Title: Activated Abl kinase inhibits oncogenic transforming growth factor-? signaling and tumorigenesis in mammary tumors

    Journal: The FASEB Journal

    doi: 10.1096/fj.09-138412

    Abl inactivation morphologically mimics EMT induced by TGF-β in normal MECs. A ) Direct actin immunofluorescence using FITC-conjugated phalloidin on Abl-manipulated NMuMG cells before and after their stimulation with TGF-β1 (5 ng/ml) or
    Figure Legend Snippet: Abl inactivation morphologically mimics EMT induced by TGF-β in normal MECs. A ) Direct actin immunofluorescence using FITC-conjugated phalloidin on Abl-manipulated NMuMG cells before and after their stimulation with TGF-β1 (5 ng/ml) or

    Techniques Used: Immunofluorescence

    39) Product Images from "Molecular Dissection of Neurobeachin Function at Excitatory Synapses"

    Article Title: Molecular Dissection of Neurobeachin Function at Excitatory Synapses

    Journal: Frontiers in Synaptic Neuroscience

    doi: 10.3389/fnsyn.2018.00028

    PH-BEACH domain restores surface targeting of GluA2 receptors in Nbea-deficient neurons. (A) Representative dendrites of primary hippocampal neurons from wild-type (WT) and Nbea null-mutant (KO) mice transfected with cytosolic marker t-dimer-RFP alone and in combination with GFP-tagged full-length Nbea (KO + Nbea FL) or the PH-BEACH domain (KO + PH-BEACH). Right panels, surface populations of GluA2 receptor subunits visualized by live labeling of neurons with an antibody directed against an extracellular epitope of the GluA2 subunit. Scale bar: 2.5 μm. (B) Quantification of surface GluA2 fluorescence intensity measured on dendrites of WT, KO, KO + Nbea FL and KO + PH-BEACH neurons. Data are means ± SEM. N , number of dendritic window/neurons (in bars); ∗∗ P
    Figure Legend Snippet: PH-BEACH domain restores surface targeting of GluA2 receptors in Nbea-deficient neurons. (A) Representative dendrites of primary hippocampal neurons from wild-type (WT) and Nbea null-mutant (KO) mice transfected with cytosolic marker t-dimer-RFP alone and in combination with GFP-tagged full-length Nbea (KO + Nbea FL) or the PH-BEACH domain (KO + PH-BEACH). Right panels, surface populations of GluA2 receptor subunits visualized by live labeling of neurons with an antibody directed against an extracellular epitope of the GluA2 subunit. Scale bar: 2.5 μm. (B) Quantification of surface GluA2 fluorescence intensity measured on dendrites of WT, KO, KO + Nbea FL and KO + PH-BEACH neurons. Data are means ± SEM. N , number of dendritic window/neurons (in bars); ∗∗ P

    Techniques Used: Mutagenesis, Mouse Assay, Transfection, Marker, Labeling, Fluorescence

    40) Product Images from "SH3 domain recognition of a proline-independent tyrosine-based RKxxYxxY motif in immune cell adaptor SKAP55"

    Article Title: SH3 domain recognition of a proline-independent tyrosine-based RKxxYxxY motif in immune cell adaptor SKAP55

    Journal: The EMBO Journal

    doi: 10.1093/emboj/19.12.2889

    Fig. 2. Key residues are required for RKGDTASYY motif binding to the FYB SH3 domain. ( A ) Amino acid sequence of peptides corresponding to SK1–SK4. The SK4 region, carrying the RKGDYASY motif, is compared with a classic class I RxxPxxP. ( B , upper panel) The SKAP55 peptide (TRRKGDYASYYQG; residues 288–300) used in precipitation studies. Various sequential mutants were synthesized in which alanine was used to substitute for amino acids. (Middle panel) AminoLink-Plus coupled to various peptides was used to precipitate the GST-tagged FYB SH3 domain from lysates from COS-1 cells. GST–FYB SH3 domain was detected by anti-GST immunoblotting. Lane 1, bovine serum albumin; lane 2, wild-type TRAKGDYASYYQG peptide; lane 3, TARKGDYASYYQG A-1 peptide; lane 4, TRAKGDYASYYQG A-2 peptide; lane 5, TRRAGDYASYYQG A-3 peptide;; lane 6, TRRKADYASYYQG A-4 peptide; lane 7, TRRKGDAASYYQG A-5 peptide; lane 8, TRRKGDYAAYYQG A-6 peptide; lane 9, TRRKGDYASAYQG A-7 peptide; lane 10, TRRKGDYASYAQG A-8 peptide; lane 11, TRRKGDYASYYAG A-9 peptide; lane 12, TRRKGDYASYYQA A-10 peptide. (Lower panel) Densitometric profile of SKAP55 in GST–FYB SH3 precipitates using a Scantjet laser scanner (Hewlett-Packard). ( C ) (Upper panel) Mutation of tyrosine residues Y294F/Y297F attenuates in vivo SKAP55 binding to the FYB SH3 domain. COS-1 cells were transfected with SKAP55, SKAP55(Y294F), SKAP55(Y297F) and FYB SH3 domain and assessed for complex formation. Glutathione–Sepharose beads were used to precipitate the GST-tagged FYB SH3 domain. Co-precipitated HA-tagged SKAP55 was detected by anti-HA immunoblotting. Lane 1, pEBG; lane 2, pEBG plus SKAP55; lane 3, SKAP55 plus FYB SH3; lane 4, SKAP55 (Y294F) plus FYB SH3; lane 5, SKAP55 (Y294F) plus FYB SH3 domain. The precipitates were separated on a 10% SDS–polyacrylamide gel and subjected to anti-HA blotting. (Middle panel) Levels of SKAP-HA protein expression. Cell lysates were separated by SDS–PAGE, transferred to nitrocellulose and subjected to blotting with anti-HA. (Lower panel) Levels of GST fusion protein expression. As in middle panel, except that lysates were blotted with anti-GST.
    Figure Legend Snippet: Fig. 2. Key residues are required for RKGDTASYY motif binding to the FYB SH3 domain. ( A ) Amino acid sequence of peptides corresponding to SK1–SK4. The SK4 region, carrying the RKGDYASY motif, is compared with a classic class I RxxPxxP. ( B , upper panel) The SKAP55 peptide (TRRKGDYASYYQG; residues 288–300) used in precipitation studies. Various sequential mutants were synthesized in which alanine was used to substitute for amino acids. (Middle panel) AminoLink-Plus coupled to various peptides was used to precipitate the GST-tagged FYB SH3 domain from lysates from COS-1 cells. GST–FYB SH3 domain was detected by anti-GST immunoblotting. Lane 1, bovine serum albumin; lane 2, wild-type TRAKGDYASYYQG peptide; lane 3, TARKGDYASYYQG A-1 peptide; lane 4, TRAKGDYASYYQG A-2 peptide; lane 5, TRRAGDYASYYQG A-3 peptide;; lane 6, TRRKADYASYYQG A-4 peptide; lane 7, TRRKGDAASYYQG A-5 peptide; lane 8, TRRKGDYAAYYQG A-6 peptide; lane 9, TRRKGDYASAYQG A-7 peptide; lane 10, TRRKGDYASYAQG A-8 peptide; lane 11, TRRKGDYASYYAG A-9 peptide; lane 12, TRRKGDYASYYQA A-10 peptide. (Lower panel) Densitometric profile of SKAP55 in GST–FYB SH3 precipitates using a Scantjet laser scanner (Hewlett-Packard). ( C ) (Upper panel) Mutation of tyrosine residues Y294F/Y297F attenuates in vivo SKAP55 binding to the FYB SH3 domain. COS-1 cells were transfected with SKAP55, SKAP55(Y294F), SKAP55(Y297F) and FYB SH3 domain and assessed for complex formation. Glutathione–Sepharose beads were used to precipitate the GST-tagged FYB SH3 domain. Co-precipitated HA-tagged SKAP55 was detected by anti-HA immunoblotting. Lane 1, pEBG; lane 2, pEBG plus SKAP55; lane 3, SKAP55 plus FYB SH3; lane 4, SKAP55 (Y294F) plus FYB SH3; lane 5, SKAP55 (Y294F) plus FYB SH3 domain. The precipitates were separated on a 10% SDS–polyacrylamide gel and subjected to anti-HA blotting. (Middle panel) Levels of SKAP-HA protein expression. Cell lysates were separated by SDS–PAGE, transferred to nitrocellulose and subjected to blotting with anti-HA. (Lower panel) Levels of GST fusion protein expression. As in middle panel, except that lysates were blotted with anti-GST.

    Techniques Used: Binding Assay, Sequencing, Synthesized, Mutagenesis, In Vivo, Transfection, Expressing, SDS Page

    Fig. 1. FYB SH3 domain binding to the SK4 region of SKAP55. ( A ) The scheme shows mouse full-length FYB, sub-regions and GST–SH3 domain constructs. (Upper panel of gel) FYB SH3 domain binds full-length SKAP55 in COS-1 cells. COS-1 cells were transfected with full-length SKAP55 alone, or with the GRB-2 or various FYB SH3 domain(s) and assessed for complex formation. Glutathione beads were used to precipitate the GST fusion proteins. Lane 1, pEBG; lane 2, pEBG and SKAP55-HA; lane 3, SKAP55-HA; lane 4, GRB-2 N-terminal SH3 domain; lane 5, GRB-2 C-terminal SH3 domain; lane 6, FYB SH3(695–762); lane 7, FYB SH3(695–770); lane 8; FYB SH3(695–775); lane 9, GRB-2 N-terminal SH3 domain and SKAP55-HA; lane 10, GRB-2 C-terminal SH3 domain and SKAP55-HA; lane 11, FYB SH3(695–762) and SKAP55-HA; lane 12, FYB SH3(695–770) and SKAP55-HA; lane 13, FYB SH3(695–775) and SKAP55-HA. The precipitates were separated on a 10% SDS–polyacrylamide gel followed by anti-HA blotting. (Middle panel) Levels of SKAP55 protein expression. As in upper panel, except cell lysate was blotted with anti-HA. (Lower panel) Levels of GST fusion protein expression. As in upper panel, except that precipitates were blotted with anti-GST. ( B ) Schematic representation of SKAP55 and various sub-regions. Various DNA fragments encoding the SKAP55 were generated by PCR and inserted into the pSRα at the Bam HI and Kpn I sites with an in-frame HA tag. (Upper panel of gel) In vivo association of FYB SH3 and SKAP55 at SK4 region. COS-1 cells were co-transfected with different GST–SKAP55 subdomains and FYB SH3 domain and assessed for complex formation. Glutathione–Sepharose beads were used to precipitate the GST fusion proteins. Lane 1, pSRαHa plus FYB SH3; lane 2, pSRαHa-SKAP55(1–106) plus FYB SH3; lane 3, pSRαHa SKAP55(105–208) plus FYB SH3; lane 4, pSRαHa-SKAP55(204–299) plus FYB SH3; lane 5, pSRα Ha-SKAP55(300–369) plus FYB SH3. The precipitates were separated on a 10% SDS–polyacrylamide gel and immunoblotted with anti-HA monoclonal antibody. (Middle panel) Levels of FYB protein expression. As in upper panel except that the cell lysate was blotted with anti-HA. (Lower panel) Levels of GST fusion protein expression. As in upper panel except that precipitates were blotted with anti-GST. ( C ) (Above) In vitro association of FYB SH3 and SKAP55 at SK4 region. The different truncated GST–SKAP55 fusion proteins used in the far-western assay. Lane 1, pGEX-SKAP55(1–106); lane 2, pGEX-SKAP55(105–208); lane 3, pGEX-SKAP55(204–299); lane 4, pGEX-SKAP55(300–369); lane 5, pGEX-SK1(204–238); lane 6, pGEX-SK2(239–255); lane 7, pGEX-SK3(256–276); lane 8, pGEX-SK4(277–298). The different truncated GST–SKAP55 fusion proteins were separated on a 10% SDS–polyacrylamide gel, transferred to nitrocellulose membranes and probed with Flag-tagged FYB SH3 protein and immunoblotted with anti-Flag monoclonal antibody. The sequence of the motifs contained within the GST fusion proteins is as follows: SK1, 204 QISFLLKDLSSLTIPYEEDEEEEEKEETYDDIDGF 238 ; SK2, 239 DSPSCGSQCRPTILPGS 255 ; SK3, 256 VGIKEPTEEKEEEDIYEVLPD 276 ; SK4, 277 EEHDLEEDESGTRRKGDYASYY 298 . (Lower panel) FYB SH3 domain recognizes the SK4 region of SKAP55. The various GST–SH2 and –SH3 fusion proteins used in the far-western assay. Lane 1, pGEX-SK4; lane 2, CRKL SH3; lane 3, LCK SH3; lane 4, p85 SH3; lane 5, Src SH3; lane 6, ABL SH2; lane 7, GRB-2 N SH2; lane 8, FYN, SH2; lane 9, LCK SH2; lane 10, PLCγ SH2-N.
    Figure Legend Snippet: Fig. 1. FYB SH3 domain binding to the SK4 region of SKAP55. ( A ) The scheme shows mouse full-length FYB, sub-regions and GST–SH3 domain constructs. (Upper panel of gel) FYB SH3 domain binds full-length SKAP55 in COS-1 cells. COS-1 cells were transfected with full-length SKAP55 alone, or with the GRB-2 or various FYB SH3 domain(s) and assessed for complex formation. Glutathione beads were used to precipitate the GST fusion proteins. Lane 1, pEBG; lane 2, pEBG and SKAP55-HA; lane 3, SKAP55-HA; lane 4, GRB-2 N-terminal SH3 domain; lane 5, GRB-2 C-terminal SH3 domain; lane 6, FYB SH3(695–762); lane 7, FYB SH3(695–770); lane 8; FYB SH3(695–775); lane 9, GRB-2 N-terminal SH3 domain and SKAP55-HA; lane 10, GRB-2 C-terminal SH3 domain and SKAP55-HA; lane 11, FYB SH3(695–762) and SKAP55-HA; lane 12, FYB SH3(695–770) and SKAP55-HA; lane 13, FYB SH3(695–775) and SKAP55-HA. The precipitates were separated on a 10% SDS–polyacrylamide gel followed by anti-HA blotting. (Middle panel) Levels of SKAP55 protein expression. As in upper panel, except cell lysate was blotted with anti-HA. (Lower panel) Levels of GST fusion protein expression. As in upper panel, except that precipitates were blotted with anti-GST. ( B ) Schematic representation of SKAP55 and various sub-regions. Various DNA fragments encoding the SKAP55 were generated by PCR and inserted into the pSRα at the Bam HI and Kpn I sites with an in-frame HA tag. (Upper panel of gel) In vivo association of FYB SH3 and SKAP55 at SK4 region. COS-1 cells were co-transfected with different GST–SKAP55 subdomains and FYB SH3 domain and assessed for complex formation. Glutathione–Sepharose beads were used to precipitate the GST fusion proteins. Lane 1, pSRαHa plus FYB SH3; lane 2, pSRαHa-SKAP55(1–106) plus FYB SH3; lane 3, pSRαHa SKAP55(105–208) plus FYB SH3; lane 4, pSRαHa-SKAP55(204–299) plus FYB SH3; lane 5, pSRα Ha-SKAP55(300–369) plus FYB SH3. The precipitates were separated on a 10% SDS–polyacrylamide gel and immunoblotted with anti-HA monoclonal antibody. (Middle panel) Levels of FYB protein expression. As in upper panel except that the cell lysate was blotted with anti-HA. (Lower panel) Levels of GST fusion protein expression. As in upper panel except that precipitates were blotted with anti-GST. ( C ) (Above) In vitro association of FYB SH3 and SKAP55 at SK4 region. The different truncated GST–SKAP55 fusion proteins used in the far-western assay. Lane 1, pGEX-SKAP55(1–106); lane 2, pGEX-SKAP55(105–208); lane 3, pGEX-SKAP55(204–299); lane 4, pGEX-SKAP55(300–369); lane 5, pGEX-SK1(204–238); lane 6, pGEX-SK2(239–255); lane 7, pGEX-SK3(256–276); lane 8, pGEX-SK4(277–298). The different truncated GST–SKAP55 fusion proteins were separated on a 10% SDS–polyacrylamide gel, transferred to nitrocellulose membranes and probed with Flag-tagged FYB SH3 protein and immunoblotted with anti-Flag monoclonal antibody. The sequence of the motifs contained within the GST fusion proteins is as follows: SK1, 204 QISFLLKDLSSLTIPYEEDEEEEEKEETYDDIDGF 238 ; SK2, 239 DSPSCGSQCRPTILPGS 255 ; SK3, 256 VGIKEPTEEKEEEDIYEVLPD 276 ; SK4, 277 EEHDLEEDESGTRRKGDYASYY 298 . (Lower panel) FYB SH3 domain recognizes the SK4 region of SKAP55. The various GST–SH2 and –SH3 fusion proteins used in the far-western assay. Lane 1, pGEX-SK4; lane 2, CRKL SH3; lane 3, LCK SH3; lane 4, p85 SH3; lane 5, Src SH3; lane 6, ABL SH2; lane 7, GRB-2 N SH2; lane 8, FYN, SH2; lane 9, LCK SH2; lane 10, PLCγ SH2-N.

    Techniques Used: Binding Assay, Construct, Transfection, Expressing, Generated, Polymerase Chain Reaction, In Vivo, In Vitro, Western Blot, Sequencing

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