gfp expression control vector  (Thermo Fisher)


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    Name:
    pLenti6 2 GW EmGFP Expression Control Vector
    Description:
    The Vivid Colors pLenti6 2 GW⁄EmGFP Expression Control Vector is a ViraPower positive control lentiviral vector containing Emerald Green Fluorescent Protein EmGFP It is designed for use with the ViraPower Lentiviral Expression Systems as a positive control to enable the detection of EmGFP fluorescence following transfection in 293FT cells as a titer control to produce an EmGFP expressing lentivirus stock and as a transduction control following transduction in both dividing and non dividing mammalian cells The vector has the CMV promoter for driving constitutive expression of EmGFP and the PGK promoter for driving long term persistent expression of the Blasticidin stable selection marker This is not a cloning vector Advantages• Optimization of viral transduction efficiency• Optimization of 293FT Transfection• 2 day titer of functional virus using EmGFPKey Features• Human cytomegalovirus CMV immediate early promoter to control high level expression of the EmGFP• PKG Promoter for expression of Blasticidin selection markerKit IncludespLenti6 2 GW⁄EmGFP VectorRelated SKUs• 293FT Cell Line R70007 R70007 • ViraPower Lentiviral Support Kit K4970 00 • ViraPower Lentiviral Packaging Mix K4975 00 • Lipofectamine 2000 11668 019 11668 027 For research use only Not intended for any therapeutic or diagnostic use
    Catalog Number:
    v36920
    Price:
    None
    Applications:
    Cell Analysis|Cell-Based Reporter Assays|Cellular Imaging|Cloning|Constitutive Expression|Destination Vectors|Enzyme Reporter Assays|Fluorescent Protein (e.g. GFP) Assays|Fluorescent Protein Assays (GFP)|Gateway Cloning|Mammalian Expression|Protein Assays and Analysis|Protein Biology|Protein Expression|Viral Gene Delivery
    Category:
    DNA Vectors Clones Purified Nucleic Acids Libraries
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    Structured Review

    Thermo Fisher gfp expression control vector
    The Vivid Colors pLenti6 2 GW⁄EmGFP Expression Control Vector is a ViraPower positive control lentiviral vector containing Emerald Green Fluorescent Protein EmGFP It is designed for use with the ViraPower Lentiviral Expression Systems as a positive control to enable the detection of EmGFP fluorescence following transfection in 293FT cells as a titer control to produce an EmGFP expressing lentivirus stock and as a transduction control following transduction in both dividing and non dividing mammalian cells The vector has the CMV promoter for driving constitutive expression of EmGFP and the PGK promoter for driving long term persistent expression of the Blasticidin stable selection marker This is not a cloning vector Advantages• Optimization of viral transduction efficiency• Optimization of 293FT Transfection• 2 day titer of functional virus using EmGFPKey Features• Human cytomegalovirus CMV immediate early promoter to control high level expression of the EmGFP• PKG Promoter for expression of Blasticidin selection markerKit IncludespLenti6 2 GW⁄EmGFP VectorRelated SKUs• 293FT Cell Line R70007 R70007 • ViraPower Lentiviral Support Kit K4970 00 • ViraPower Lentiviral Packaging Mix K4975 00 • Lipofectamine 2000 11668 019 11668 027 For research use only Not intended for any therapeutic or diagnostic use
    https://www.bioz.com/result/gfp expression control vector/product/Thermo Fisher
    Average 92 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    gfp expression control vector - by Bioz Stars, 2020-08
    92/100 stars

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    gfp transcript copies
    gfp dna

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    Produced:

    Article Title: Co-culture of bone marrow stromal cells and chondrocytes in vivo for the repair of the goat condylar cartilage defects
    Article Snippet: .. GFP lentiviruses were produced by transient transfection into the 293FT cell line (Invitrogen; Thermo Fisher Scientific, Inc.) with the pLenti6.2-GW/EmGFP vector and three packaging plasmids, pLP1, pLP2, and pLP/VSVG (Invitrogen; Thermo Fisher Scientific, Inc.). .. For the isolation of auricular chondrocytes, auricular cartilage (3×3 cm2 ) was harvested from the goat ear by a sterile surgical technique and cut into 2×2 mm2 sections.

    Plasmid Purification:

    Article Title: Co-culture of bone marrow stromal cells and chondrocytes in vivo for the repair of the goat condylar cartilage defects
    Article Snippet: .. GFP lentiviruses were produced by transient transfection into the 293FT cell line (Invitrogen; Thermo Fisher Scientific, Inc.) with the pLenti6.2-GW/EmGFP vector and three packaging plasmids, pLP1, pLP2, and pLP/VSVG (Invitrogen; Thermo Fisher Scientific, Inc.). .. For the isolation of auricular chondrocytes, auricular cartilage (3×3 cm2 ) was harvested from the goat ear by a sterile surgical technique and cut into 2×2 mm2 sections.

    Transfection:

    Article Title: Co-culture of bone marrow stromal cells and chondrocytes in vivo for the repair of the goat condylar cartilage defects
    Article Snippet: .. GFP lentiviruses were produced by transient transfection into the 293FT cell line (Invitrogen; Thermo Fisher Scientific, Inc.) with the pLenti6.2-GW/EmGFP vector and three packaging plasmids, pLP1, pLP2, and pLP/VSVG (Invitrogen; Thermo Fisher Scientific, Inc.). .. For the isolation of auricular chondrocytes, auricular cartilage (3×3 cm2 ) was harvested from the goat ear by a sterile surgical technique and cut into 2×2 mm2 sections.

    Plasmid Preparation:

    Article Title: Functionally competent eosinophils differentiated ex vivo in high purity from normal mouse bone marrow
    Article Snippet: .. The Vivid Colors pLenti6.2GW/EmGFP vector and the Virapower expression system (Invitrogen) was used to infect 293FT cells per manufacturer's instructions. .. The resulting viral supernatant was collected and concentrated 100-fold by ultracentrifugation, and the viral titer was determined using a p24 ELISA kit (Cell-Biolabs).

    Article Title: Co-culture of bone marrow stromal cells and chondrocytes in vivo for the repair of the goat condylar cartilage defects
    Article Snippet: .. GFP lentiviruses were produced by transient transfection into the 293FT cell line (Invitrogen; Thermo Fisher Scientific, Inc.) with the pLenti6.2-GW/EmGFP vector and three packaging plasmids, pLP1, pLP2, and pLP/VSVG (Invitrogen; Thermo Fisher Scientific, Inc.). .. For the isolation of auricular chondrocytes, auricular cartilage (3×3 cm2 ) was harvested from the goat ear by a sterile surgical technique and cut into 2×2 mm2 sections.

    Article Title: Intracoronary Administration of Allogeneic Adipose Tissue–Derived Mesenchymal Stem Cells Improves Myocardial Perfusion But Not Left Ventricle Function, in a Translational Model of Acute Myocardial Infarction
    Article Snippet: .. Number of GFP transcript copies was established by comparison to a calibration curve based on known amounts of GFP DNA from GFP expression control vector (pLenti6.2‐GW/EmGFP; Invitrogen, Waltham, MA). .. The standard curve for GFP DNA was obtained by 10‐fold serial dilution from 1 ng (3×106 copy number of GFP) to 1 fg (3 copy number of GFP) of the GFP expression control vector (Figure ).

    Expressing:

    Article Title: Functionally competent eosinophils differentiated ex vivo in high purity from normal mouse bone marrow
    Article Snippet: .. The Vivid Colors pLenti6.2GW/EmGFP vector and the Virapower expression system (Invitrogen) was used to infect 293FT cells per manufacturer's instructions. .. The resulting viral supernatant was collected and concentrated 100-fold by ultracentrifugation, and the viral titer was determined using a p24 ELISA kit (Cell-Biolabs).

    Article Title: Intracoronary Administration of Allogeneic Adipose Tissue–Derived Mesenchymal Stem Cells Improves Myocardial Perfusion But Not Left Ventricle Function, in a Translational Model of Acute Myocardial Infarction
    Article Snippet: .. Number of GFP transcript copies was established by comparison to a calibration curve based on known amounts of GFP DNA from GFP expression control vector (pLenti6.2‐GW/EmGFP; Invitrogen, Waltham, MA). .. The standard curve for GFP DNA was obtained by 10‐fold serial dilution from 1 ng (3×106 copy number of GFP) to 1 fg (3 copy number of GFP) of the GFP expression control vector (Figure ).

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    Thermo Fisher gfp expressing control vector gfp
    APP binds the MA region of HIV-1 Gag. a <t>GFP-tagged</t> human APP 770 (GFP-APP), but not GFP control, binds HIV-1 Gag (Gag-HA) or Matrix (MA-HA), but not Capsid (CA-HA) in GBP-binding assays. b Endogenous APP interacts with Pr55 Gag in WT HIV-1-infected CHME3 4 × 4 cells in anti-APP co-IP. *indicates unspecific bands detected in cell lysates. c Schematic of the HIV-1 Gag polyprotein used in binding assays, including the c-terminal HA tag. X indicates a point mutation in the N-terminal myristoylation site (Gag-N-Myr-HA). Sequential 20 aa deletions are indicated. d Gag mutants lacking aa 72-111 of MA (Gag-MA-40-HA, Gag-MA-60-HA, or Gag-MA-80-HA) fail to bind GFP-APP in <t>co-transfected</t> 293T cells in GBP-binding assay. e Gag mutants lacking aa 72-111 of MA (Gag-MA-40-HA, Gag-MA-60-HA, or Gag-MA-80-HA) fail to bind endogenous APP in CHME3 cells in anti-APP co-IP. Molecular weight markers (in kDa) are shown to the right of WBs
    Gfp Expressing Control Vector Gfp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gfp expressing control vector gfp/product/Thermo Fisher
    Average 91 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    gfp expressing control vector gfp - by Bioz Stars, 2020-08
    91/100 stars
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    84
    Thermo Fisher plenti6 2 gw emgfp expression control vector
    APP binds the MA region of HIV-1 Gag. a <t>GFP-tagged</t> human APP 770 (GFP-APP), but not GFP control, binds HIV-1 Gag (Gag-HA) or Matrix (MA-HA), but not Capsid (CA-HA) in GBP-binding assays. b Endogenous APP interacts with Pr55 Gag in WT HIV-1-infected CHME3 4 × 4 cells in anti-APP co-IP. *indicates unspecific bands detected in cell lysates. c Schematic of the HIV-1 Gag polyprotein used in binding assays, including the c-terminal HA tag. X indicates a point mutation in the N-terminal myristoylation site (Gag-N-Myr-HA). Sequential 20 aa deletions are indicated. d Gag mutants lacking aa 72-111 of MA (Gag-MA-40-HA, Gag-MA-60-HA, or Gag-MA-80-HA) fail to bind GFP-APP in <t>co-transfected</t> 293T cells in GBP-binding assay. e Gag mutants lacking aa 72-111 of MA (Gag-MA-40-HA, Gag-MA-60-HA, or Gag-MA-80-HA) fail to bind endogenous APP in CHME3 cells in anti-APP co-IP. Molecular weight markers (in kDa) are shown to the right of WBs
    Plenti6 2 Gw Emgfp Expression Control Vector, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/plenti6 2 gw emgfp expression control vector/product/Thermo Fisher
    Average 84 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    plenti6 2 gw emgfp expression control vector - by Bioz Stars, 2020-08
    84/100 stars
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    APP binds the MA region of HIV-1 Gag. a GFP-tagged human APP 770 (GFP-APP), but not GFP control, binds HIV-1 Gag (Gag-HA) or Matrix (MA-HA), but not Capsid (CA-HA) in GBP-binding assays. b Endogenous APP interacts with Pr55 Gag in WT HIV-1-infected CHME3 4 × 4 cells in anti-APP co-IP. *indicates unspecific bands detected in cell lysates. c Schematic of the HIV-1 Gag polyprotein used in binding assays, including the c-terminal HA tag. X indicates a point mutation in the N-terminal myristoylation site (Gag-N-Myr-HA). Sequential 20 aa deletions are indicated. d Gag mutants lacking aa 72-111 of MA (Gag-MA-40-HA, Gag-MA-60-HA, or Gag-MA-80-HA) fail to bind GFP-APP in co-transfected 293T cells in GBP-binding assay. e Gag mutants lacking aa 72-111 of MA (Gag-MA-40-HA, Gag-MA-60-HA, or Gag-MA-80-HA) fail to bind endogenous APP in CHME3 cells in anti-APP co-IP. Molecular weight markers (in kDa) are shown to the right of WBs

    Journal: Nature Communications

    Article Title: HIV-1 counteracts an innate restriction by amyloid precursor protein resulting in neurodegeneration

    doi: 10.1038/s41467-017-01795-8

    Figure Lengend Snippet: APP binds the MA region of HIV-1 Gag. a GFP-tagged human APP 770 (GFP-APP), but not GFP control, binds HIV-1 Gag (Gag-HA) or Matrix (MA-HA), but not Capsid (CA-HA) in GBP-binding assays. b Endogenous APP interacts with Pr55 Gag in WT HIV-1-infected CHME3 4 × 4 cells in anti-APP co-IP. *indicates unspecific bands detected in cell lysates. c Schematic of the HIV-1 Gag polyprotein used in binding assays, including the c-terminal HA tag. X indicates a point mutation in the N-terminal myristoylation site (Gag-N-Myr-HA). Sequential 20 aa deletions are indicated. d Gag mutants lacking aa 72-111 of MA (Gag-MA-40-HA, Gag-MA-60-HA, or Gag-MA-80-HA) fail to bind GFP-APP in co-transfected 293T cells in GBP-binding assay. e Gag mutants lacking aa 72-111 of MA (Gag-MA-40-HA, Gag-MA-60-HA, or Gag-MA-80-HA) fail to bind endogenous APP in CHME3 cells in anti-APP co-IP. Molecular weight markers (in kDa) are shown to the right of WBs

    Article Snippet: For GBP-binding assay, 293T cells (3 × 106 ) were transfected with 2 μg of a GFP-expressing control vector (GFP) or N′-GFP-APP770 (GFP-APP) along with 2 μg of HA-tagged forms of HIV-1 Gag (Gag-HA, Gag-N-Myr-HA or Gag-MA: -20-HA, -40-HA, -60-HA, and -80-HA) using 15 μl TurboFect (Thermo scientific).

    Techniques: Binding Assay, Infection, Co-Immunoprecipitation Assay, Mutagenesis, Transfection, Molecular Weight

    Binding to kinesin-1 is required for FEZ1 to promote early HIV-1 infection (a) 293A cells were transfected with vectors expressing a control GFP or GFP-Kif5B tail along with either FEZ1-Flag or FEZ1-S58A-Flag (S58A-Flag) constructs. Soluble cell extracts were prepared 48h post-transfection, precleared and GFP proteins were recovered by incubating samples with GFP-binding protein (GBP) covalently coupled to Sepharose. Input and bound proteins were then analyzed by WB using anti-Flag and anti-GFP antibodies. (b) Pools of NHDF cells stably expressing Flag control (Control), full-length C-terminally flag-tagged FEZ1 (FEZ1-Flag) or a C-terminally flag-tagged FEZ1 mutant unable to bind kinesin-1 (S58A-Flag) were lysed and analyzed by WB using antibodies against Flag or β-actin (loading control). (c–d) NHDF pools described in b. were infected with HIV-1-VSV-luc (c) or HIV-1-Ampho-luc (d) followed by measurements of luciferase activity 48h post-infection to determine levels of infection. Results are representative of 3 or more independent experiments, and error bars represent standard deviation. (e) Binding of FEZ1 or FEZ1-S58A to in vitro assembled HIV-1 CA-NC complexes. 293T cells were transfected with FEZ1-Flag, FEZ1-S58A-Flag (S58a-Flag) or NES-CPSF6-Flag constructs as described in the legend for Fig. 2 . Cells were lysed 36h post-transfection and the lysates were incubated at room temperature for 1h with in vitro assembled HIV-1 CA-NC complexes. The lysates were then analyzed by WB either before (Input) or after sedimentation through a 70% sucrose cushion (Bound) using anti-Flag and anti-p24 antibodies. (f) Binding of FEZ1-S58A to HIV-1 Gag by co-IP. 293A cells were co-transfected with a HA-tagged HIV-1 Gag expressing vector alone or together with FEZ1-Flag or FEZ1-S58A-Flag (S58A-Flag). Cells were lysed 48h post-transfection, and proteins were co-immunoprecipitated using anti-HA antibodies. Input and bound samples were then analyzed by WB either before (Input) or after immunoprecipitation (IP) using anti-Flag and anti-HA antibodies. Molecular weight markers (in kDa) are shown to the right of WBs.

    Journal: Nature communications

    Article Title: HIV-1 capsids bind and exploit the kinesin-1 adaptor FEZ1 for inward movement to the nucleus

    doi: 10.1038/ncomms7660

    Figure Lengend Snippet: Binding to kinesin-1 is required for FEZ1 to promote early HIV-1 infection (a) 293A cells were transfected with vectors expressing a control GFP or GFP-Kif5B tail along with either FEZ1-Flag or FEZ1-S58A-Flag (S58A-Flag) constructs. Soluble cell extracts were prepared 48h post-transfection, precleared and GFP proteins were recovered by incubating samples with GFP-binding protein (GBP) covalently coupled to Sepharose. Input and bound proteins were then analyzed by WB using anti-Flag and anti-GFP antibodies. (b) Pools of NHDF cells stably expressing Flag control (Control), full-length C-terminally flag-tagged FEZ1 (FEZ1-Flag) or a C-terminally flag-tagged FEZ1 mutant unable to bind kinesin-1 (S58A-Flag) were lysed and analyzed by WB using antibodies against Flag or β-actin (loading control). (c–d) NHDF pools described in b. were infected with HIV-1-VSV-luc (c) or HIV-1-Ampho-luc (d) followed by measurements of luciferase activity 48h post-infection to determine levels of infection. Results are representative of 3 or more independent experiments, and error bars represent standard deviation. (e) Binding of FEZ1 or FEZ1-S58A to in vitro assembled HIV-1 CA-NC complexes. 293T cells were transfected with FEZ1-Flag, FEZ1-S58A-Flag (S58a-Flag) or NES-CPSF6-Flag constructs as described in the legend for Fig. 2 . Cells were lysed 36h post-transfection and the lysates were incubated at room temperature for 1h with in vitro assembled HIV-1 CA-NC complexes. The lysates were then analyzed by WB either before (Input) or after sedimentation through a 70% sucrose cushion (Bound) using anti-Flag and anti-p24 antibodies. (f) Binding of FEZ1-S58A to HIV-1 Gag by co-IP. 293A cells were co-transfected with a HA-tagged HIV-1 Gag expressing vector alone or together with FEZ1-Flag or FEZ1-S58A-Flag (S58A-Flag). Cells were lysed 48h post-transfection, and proteins were co-immunoprecipitated using anti-HA antibodies. Input and bound samples were then analyzed by WB either before (Input) or after immunoprecipitation (IP) using anti-Flag and anti-HA antibodies. Molecular weight markers (in kDa) are shown to the right of WBs.

    Article Snippet: The next day, cells were co-transfected with equimolar amounts of FEZ1-Flag or FEZ1-S58A-Flag expressing vectors along with either a GFP expressing control vector or a construct encoding a GFP-tagged Kif5B-tail domain using 28μl Turbofect (Thermo Scientific) as per manufacturers instructions.

    Techniques: Binding Assay, Infection, Transfection, Expressing, Construct, Western Blot, Stable Transfection, Mutagenesis, Luciferase, Activity Assay, Standard Deviation, In Vitro, Incubation, Sedimentation, Co-Immunoprecipitation Assay, Plasmid Preparation, Immunoprecipitation, Molecular Weight

    Depletion of FEZ1 inhibits HIV-1 trafficking to the nucleus NHDFs were treated with control or FEZ1-C siRNAs. 48h post-transfection cells were infected with HIV-1-VSV-GFP-Vpr followed by live imaging using a spinning-disc confocal microscope. (a) Still images from movies ( Supplementary Movies 1 and 2 ) taken at the indicated times are shown. Green arrows highlight viral particles entering and traversing the cytoplasm in control siRNA-treated cells. Red arrows highlight representative examples of particles that approach the nucleus but then move long distances back out to the cell periphery in FEZ1-depleted cells. (b) Quantification of the average distance (μm per 2.5 min) traveled by viral particles towards the nucleus (Retrograde motility) or away from the nucleus (Antrograde motility). n≥5 cells and an average of 7–30 viral particles per cell. (c) Quantification of the percentage of virions within 2μm of the nucleus in infected siRNA-treated cells at the indicated time points. n≥20 cells and an average of 80–99 viral particles per cell. (d–f) Depletion of FEZ1 affects HIV-1 particles that have productively fused into the cytoplasm. (d) NHDF cells were treated with control or FEZ1-C siRNAs. 48h after transfection cells were infected with HIV-1-VSV containing GFP-Vpr and S15-Tomato. 1h post-infection cells were fixed in formaldehyde and GFP and Tomato signals were acquired using a spinning-disc confocal microscope. Arrows highlight fused (green, S15-negative) particles proximal to the nucleus in each sample. Representative confocal planes are shown. (e) Quantification of the % fused (green, S15-negative) viral particles within 2μm of the nucleus in samples as described and processed in d. n≥29 cells and an average of 53–55 viral particles per cell. (f) Control siRNA-treated NHDF cells were treated with Bafilomycin A1 for 2h during spinoculation followed by infection with HIV-1-VSV containing GFP-Vpr and S15-Tomato. 1h post-infection cells were fixed and the total number of fused (green, S15-negative) viral particles were quantified and presented as a % of the total number of viral particles. n≥29 cells and an average of 55–80 viral particles per cell. Error bars represent standard deviation. Scale bars represent 10 μm.

    Journal: Nature communications

    Article Title: HIV-1 capsids bind and exploit the kinesin-1 adaptor FEZ1 for inward movement to the nucleus

    doi: 10.1038/ncomms7660

    Figure Lengend Snippet: Depletion of FEZ1 inhibits HIV-1 trafficking to the nucleus NHDFs were treated with control or FEZ1-C siRNAs. 48h post-transfection cells were infected with HIV-1-VSV-GFP-Vpr followed by live imaging using a spinning-disc confocal microscope. (a) Still images from movies ( Supplementary Movies 1 and 2 ) taken at the indicated times are shown. Green arrows highlight viral particles entering and traversing the cytoplasm in control siRNA-treated cells. Red arrows highlight representative examples of particles that approach the nucleus but then move long distances back out to the cell periphery in FEZ1-depleted cells. (b) Quantification of the average distance (μm per 2.5 min) traveled by viral particles towards the nucleus (Retrograde motility) or away from the nucleus (Antrograde motility). n≥5 cells and an average of 7–30 viral particles per cell. (c) Quantification of the percentage of virions within 2μm of the nucleus in infected siRNA-treated cells at the indicated time points. n≥20 cells and an average of 80–99 viral particles per cell. (d–f) Depletion of FEZ1 affects HIV-1 particles that have productively fused into the cytoplasm. (d) NHDF cells were treated with control or FEZ1-C siRNAs. 48h after transfection cells were infected with HIV-1-VSV containing GFP-Vpr and S15-Tomato. 1h post-infection cells were fixed in formaldehyde and GFP and Tomato signals were acquired using a spinning-disc confocal microscope. Arrows highlight fused (green, S15-negative) particles proximal to the nucleus in each sample. Representative confocal planes are shown. (e) Quantification of the % fused (green, S15-negative) viral particles within 2μm of the nucleus in samples as described and processed in d. n≥29 cells and an average of 53–55 viral particles per cell. (f) Control siRNA-treated NHDF cells were treated with Bafilomycin A1 for 2h during spinoculation followed by infection with HIV-1-VSV containing GFP-Vpr and S15-Tomato. 1h post-infection cells were fixed and the total number of fused (green, S15-negative) viral particles were quantified and presented as a % of the total number of viral particles. n≥29 cells and an average of 55–80 viral particles per cell. Error bars represent standard deviation. Scale bars represent 10 μm.

    Article Snippet: The next day, cells were co-transfected with equimolar amounts of FEZ1-Flag or FEZ1-S58A-Flag expressing vectors along with either a GFP expressing control vector or a construct encoding a GFP-tagged Kif5B-tail domain using 28μl Turbofect (Thermo Scientific) as per manufacturers instructions.

    Techniques: Transfection, Infection, Imaging, Microscopy, Standard Deviation

    Kinesin-1 regulates nuclear entry of HIV-1 DNA (a–g) Kinesin-1 depletion affects early HIV-1 infection regardless of the route of viral entry. NHDF (a–b), CHME3 (c–d,g) or Jurkat (e–f) cells were transfected with control (ctrl), Kif5A or Kif5B siRNAs. 48h post-transfection cells were infected with HIV-1-VSV-luc (a, c, e) or HIV-1-Ampho-luc (g) followed by measurements of luciferase activity in NHDF (a), CHME3 (c, g) or Jurkat (e) cells. (b, d and f). WB analysis demonstrated kinesin-1 depletion in samples from a, c and e using antibodies against kinesin-1 (Kif5A/B), Kif5B or β-actin (loading control). (h) Kinesin-1 knockdown does not inhibit VSV infection. NHDF cells treated with control, Kif5A or Kif5B siRNAs were infected 48h post-transfection with VSV at m.o.i. 10. 8h after infection cells were lysed and analyzed by WB using anti-VSV-G or anti-VSV-N antibodies. eIF4E was used as loading control. (i) Effects of kinesin-1 depletion on fusion of HIV-1 cores into the cytosol. NHDFs were treated with control, Kif5A or Kif5B siRNAs and then either mock infected (upper panels) or infected with HIV-1-VSV-luc containing BlaM-Vpr (lower panels). FACS analysis of cells showed ~14–18% shift from green (uncleaved CCF2) to blue (cleaved CCF2) cells in control and kinesin-1-depleted cultures. (j–l) Kinesin-1 regulates nuclear entry of HIV-1 DNA. (j) CHME3 cells treated with control (Ctrl), Kif5A or Kif5B siRNAs were infected with HIV-1-VSV-puro 48h post-transfection. Low molecular Hirt DNA was isolated 24h post-infection and levels of viral MSS-DNA, total viral DNA and 2-LTRs in samples were measured by qPCR using specific primers to MSS, puromycin or 2-LTRs, respectively. Copy numbers were calculated and normalized to input DNA in each sample. Data are presented as mean +/- SEM. (k–l) CHME3 cells were transfected with plasmids expressing either GFP or GFP-tagged dominant negative Kif5B (Kif5B-GFP-DN). 48h post-transfection cells were infected (k) and levels of MSS DNA, total DN and 2-LTRs were measured as described in j. or (l) cells were lysed in Laemmli buffer and analyzed by WB using anti-GFP antibody to detect GFP or dominant-negative GFP-Kif5 (Kif5B-GFP-DN). Molecular weight markers (in kDa) are shown to the right of WBs.

    Journal: Nature communications

    Article Title: HIV-1 capsids bind and exploit the kinesin-1 adaptor FEZ1 for inward movement to the nucleus

    doi: 10.1038/ncomms7660

    Figure Lengend Snippet: Kinesin-1 regulates nuclear entry of HIV-1 DNA (a–g) Kinesin-1 depletion affects early HIV-1 infection regardless of the route of viral entry. NHDF (a–b), CHME3 (c–d,g) or Jurkat (e–f) cells were transfected with control (ctrl), Kif5A or Kif5B siRNAs. 48h post-transfection cells were infected with HIV-1-VSV-luc (a, c, e) or HIV-1-Ampho-luc (g) followed by measurements of luciferase activity in NHDF (a), CHME3 (c, g) or Jurkat (e) cells. (b, d and f). WB analysis demonstrated kinesin-1 depletion in samples from a, c and e using antibodies against kinesin-1 (Kif5A/B), Kif5B or β-actin (loading control). (h) Kinesin-1 knockdown does not inhibit VSV infection. NHDF cells treated with control, Kif5A or Kif5B siRNAs were infected 48h post-transfection with VSV at m.o.i. 10. 8h after infection cells were lysed and analyzed by WB using anti-VSV-G or anti-VSV-N antibodies. eIF4E was used as loading control. (i) Effects of kinesin-1 depletion on fusion of HIV-1 cores into the cytosol. NHDFs were treated with control, Kif5A or Kif5B siRNAs and then either mock infected (upper panels) or infected with HIV-1-VSV-luc containing BlaM-Vpr (lower panels). FACS analysis of cells showed ~14–18% shift from green (uncleaved CCF2) to blue (cleaved CCF2) cells in control and kinesin-1-depleted cultures. (j–l) Kinesin-1 regulates nuclear entry of HIV-1 DNA. (j) CHME3 cells treated with control (Ctrl), Kif5A or Kif5B siRNAs were infected with HIV-1-VSV-puro 48h post-transfection. Low molecular Hirt DNA was isolated 24h post-infection and levels of viral MSS-DNA, total viral DNA and 2-LTRs in samples were measured by qPCR using specific primers to MSS, puromycin or 2-LTRs, respectively. Copy numbers were calculated and normalized to input DNA in each sample. Data are presented as mean +/- SEM. (k–l) CHME3 cells were transfected with plasmids expressing either GFP or GFP-tagged dominant negative Kif5B (Kif5B-GFP-DN). 48h post-transfection cells were infected (k) and levels of MSS DNA, total DN and 2-LTRs were measured as described in j. or (l) cells were lysed in Laemmli buffer and analyzed by WB using anti-GFP antibody to detect GFP or dominant-negative GFP-Kif5 (Kif5B-GFP-DN). Molecular weight markers (in kDa) are shown to the right of WBs.

    Article Snippet: The next day, cells were co-transfected with equimolar amounts of FEZ1-Flag or FEZ1-S58A-Flag expressing vectors along with either a GFP expressing control vector or a construct encoding a GFP-tagged Kif5B-tail domain using 28μl Turbofect (Thermo Scientific) as per manufacturers instructions.

    Techniques: Infection, Transfection, Luciferase, Activity Assay, Western Blot, FACS, Isolation, Real-time Polymerase Chain Reaction, Expressing, Dominant Negative Mutation, Molecular Weight

    Impact of RagC S75Y on mTORC1 signaling. A. Immunoassay showing overexpression of RagC S75Y results in increased mTORC1 signaling in the absence of amino acids as compared to wild-type RagC, as indicated by P-S6K and P-S6. AD293 cells overexpressing control-GFP

    Journal: Human genetics

    Article Title: De novo RRAGC mutation activates mTORC1 signaling in syndromic fetal dilated cardiomyopathy

    doi: 10.1007/s00439-016-1685-3

    Figure Lengend Snippet: Impact of RagC S75Y on mTORC1 signaling. A. Immunoassay showing overexpression of RagC S75Y results in increased mTORC1 signaling in the absence of amino acids as compared to wild-type RagC, as indicated by P-S6K and P-S6. AD293 cells overexpressing control-GFP

    Article Snippet: AD293 cells were transfected with plasmid constructs expressing control-GFP (empty vector), wild-type (WT) RagC, or RagCS75Y using Lipofectamine 2000 (Thermofisher).

    Techniques: Over Expression