anti ha antibody conjugated agarose beads  (Roche)


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    Structured Review

    Roche anti ha antibody conjugated agarose beads
    The D 14 RMR 17 and Trp79 domains mediate the interaction between Vif and A3DE. HIV-1 Vif DR14/15 and W79 all showed reduced interaction with A3DE when compared to WT Vif. HEK293T cells were cotransfected with A3DE and a control vector, WT Vif, or one of the indicated Vif mutants. At 48 h post-transfection, cell lysates were prepared and immunoprecipitated with <t>anti-myc</t> <t>antibody</t> and <t>agarose-conjugated</t> protein A/G. Cell lysates (A) and the interaction of A3C with WT or mutant Vif molecules(B) were detected by immunoblotting with antibodies against <t>A3DE-HA</t> and Vif-myc.
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    Images

    1) Product Images from "Distinct Determinants in HIV-1 Vif and Human APOBEC3 Proteins Are Required for the Suppression of Diverse Host Anti-Viral Proteins"

    Article Title: Distinct Determinants in HIV-1 Vif and Human APOBEC3 Proteins Are Required for the Suppression of Diverse Host Anti-Viral Proteins

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0003963

    The D 14 RMR 17 and Trp79 domains mediate the interaction between Vif and A3DE. HIV-1 Vif DR14/15 and W79 all showed reduced interaction with A3DE when compared to WT Vif. HEK293T cells were cotransfected with A3DE and a control vector, WT Vif, or one of the indicated Vif mutants. At 48 h post-transfection, cell lysates were prepared and immunoprecipitated with anti-myc antibody and agarose-conjugated protein A/G. Cell lysates (A) and the interaction of A3C with WT or mutant Vif molecules(B) were detected by immunoblotting with antibodies against A3DE-HA and Vif-myc.
    Figure Legend Snippet: The D 14 RMR 17 and Trp79 domains mediate the interaction between Vif and A3DE. HIV-1 Vif DR14/15 and W79 all showed reduced interaction with A3DE when compared to WT Vif. HEK293T cells were cotransfected with A3DE and a control vector, WT Vif, or one of the indicated Vif mutants. At 48 h post-transfection, cell lysates were prepared and immunoprecipitated with anti-myc antibody and agarose-conjugated protein A/G. Cell lysates (A) and the interaction of A3C with WT or mutant Vif molecules(B) were detected by immunoblotting with antibodies against A3DE-HA and Vif-myc.

    Techniques Used: Plasmid Preparation, Transfection, Immunoprecipitation, Mutagenesis

    The C-CCD of A3F behaves like the full-length A3F. (A) Alignment of A3C and the C-CCD of AF. (B) Alignment of A3C and the N-CCD of AF. (C) Interaction of A3F and A3F-C with HIV-1 Vif. HEK293T cells were cotransfected with HIV-1 Vif-myc plus control vector, full-length A3F-HA, or A3F-C-HA. The cells were treated with 10 µM MG132 12 h prior to harvesting., and A3F-HA proteins were immunoprecipitated from cell lysates with an anti-HA antibody conjugated to agarose beads. The interaction of A3F with HIV-1 Vif molecules was detected by immunoblotting with antibodies against A3F-HA and Vif antibody. (D) HIV-1 Vif induces the degradation of the C-CCD of A3F. HEK293T cells were transfected with an expression vector encoding the C-CCD of A3F plus a control vector, WT Vif, or one of the indicated Vif mutant expression vectors. A3F-C stability was assessed by immunoblotting with antibodies against V5, Vif-myc, and β-tubulin as a loading control.
    Figure Legend Snippet: The C-CCD of A3F behaves like the full-length A3F. (A) Alignment of A3C and the C-CCD of AF. (B) Alignment of A3C and the N-CCD of AF. (C) Interaction of A3F and A3F-C with HIV-1 Vif. HEK293T cells were cotransfected with HIV-1 Vif-myc plus control vector, full-length A3F-HA, or A3F-C-HA. The cells were treated with 10 µM MG132 12 h prior to harvesting., and A3F-HA proteins were immunoprecipitated from cell lysates with an anti-HA antibody conjugated to agarose beads. The interaction of A3F with HIV-1 Vif molecules was detected by immunoblotting with antibodies against A3F-HA and Vif antibody. (D) HIV-1 Vif induces the degradation of the C-CCD of A3F. HEK293T cells were transfected with an expression vector encoding the C-CCD of A3F plus a control vector, WT Vif, or one of the indicated Vif mutant expression vectors. A3F-C stability was assessed by immunoblotting with antibodies against V5, Vif-myc, and β-tubulin as a loading control.

    Techniques Used: Plasmid Preparation, Immunoprecipitation, Transfection, Expressing, Mutagenesis

    The D 14 RMR 17 and Trp79 domains mediate the interaction between HIV-1 Vif and A3C. Vif DR14/15 and W79 showed reduced interaction with A3C when compared to WT Vif. HEK293T cells were cotransfected with A3C and the control vector, HIV-1 Vif, or one of the indicated Vif mutants. At 48 h post-transfection, cell lysates were prepared and immunoprecipitated with anti-myc antibody and agarose-conjugated protein A/G. Cell lysate (A) and the interaction of A3C with WT or mutant Vif molecules(B) were detected by immunoblotting with antibodies against A3G-HA and Vif-myc.
    Figure Legend Snippet: The D 14 RMR 17 and Trp79 domains mediate the interaction between HIV-1 Vif and A3C. Vif DR14/15 and W79 showed reduced interaction with A3C when compared to WT Vif. HEK293T cells were cotransfected with A3C and the control vector, HIV-1 Vif, or one of the indicated Vif mutants. At 48 h post-transfection, cell lysates were prepared and immunoprecipitated with anti-myc antibody and agarose-conjugated protein A/G. Cell lysate (A) and the interaction of A3C with WT or mutant Vif molecules(B) were detected by immunoblotting with antibodies against A3G-HA and Vif-myc.

    Techniques Used: Plasmid Preparation, Transfection, Immunoprecipitation, Mutagenesis

    2) Product Images from "Self-Association of Gata1 Enhances Transcriptional Activity In Vivo in Zebra Fish Embryos"

    Article Title: Self-Association of Gata1 Enhances Transcriptional Activity In Vivo in Zebra Fish Embryos

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.23.22.8295-8305.2003

    Self-association of zebra fish Gata1 proteins. HA-tagged (lanes 1 and 2) and FLAG (FL)-tagged (lanes 3 and 4) proteins of wild-type Gata1 (WT) (lanes 1 and 3) and Gata1KA6 (lanes 2 and 4) were examined by immunoblot (IB) analysis with anti-FLAG (αFL) (upper panel) or anti-HA (lower panel) antibodies. Arrows indicate migration positions of the Gata1 proteins. Mixtures of FLAG-tagged and HA-tagged proteins (lanes 5 and 8), FLAG-tagged protein alone (lanes 6 and 9), HA-tagged protein alone (lanes 7 and 10) for wild-type Gata1 (lanes 5 to 7) or Gata1KA6 (lanes 8 to 10) were immunoprecipitated with anti-HA-conjugated agarose beads. Precipitated proteins were analyzed by immunoblotting.
    Figure Legend Snippet: Self-association of zebra fish Gata1 proteins. HA-tagged (lanes 1 and 2) and FLAG (FL)-tagged (lanes 3 and 4) proteins of wild-type Gata1 (WT) (lanes 1 and 3) and Gata1KA6 (lanes 2 and 4) were examined by immunoblot (IB) analysis with anti-FLAG (αFL) (upper panel) or anti-HA (lower panel) antibodies. Arrows indicate migration positions of the Gata1 proteins. Mixtures of FLAG-tagged and HA-tagged proteins (lanes 5 and 8), FLAG-tagged protein alone (lanes 6 and 9), HA-tagged protein alone (lanes 7 and 10) for wild-type Gata1 (lanes 5 to 7) or Gata1KA6 (lanes 8 to 10) were immunoprecipitated with anti-HA-conjugated agarose beads. Precipitated proteins were analyzed by immunoblotting.

    Techniques Used: Fluorescence In Situ Hybridization, Migration, Immunoprecipitation

    3) Product Images from "Identification of HIV-1 Vif Regions Required for CBF-? Interaction and APOBEC3 Suppression"

    Article Title: Identification of HIV-1 Vif Regions Required for CBF-? Interaction and APOBEC3 Suppression

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0095738

    Vif mutants maintain interactions with A3G and A3F. HEK293T cells were transfected with the vector control, WT or a Vif mutant plus A3G-V5 (A) or A3F-V5 (B). Cells were treated with 10 µM MG132 12 h prior to harvesting, and then Vif-HA was immunoprecipitated from cell lysates with an anti-HA antibody conjugated to agarose beads. The interaction of Vif-HA with A3G-V5 or A3F-V5 was detected by immunoblotting using antibodies against Vif-HA and A3G-V5 or A3F-V5.
    Figure Legend Snippet: Vif mutants maintain interactions with A3G and A3F. HEK293T cells were transfected with the vector control, WT or a Vif mutant plus A3G-V5 (A) or A3F-V5 (B). Cells were treated with 10 µM MG132 12 h prior to harvesting, and then Vif-HA was immunoprecipitated from cell lysates with an anti-HA antibody conjugated to agarose beads. The interaction of Vif-HA with A3G-V5 or A3F-V5 was detected by immunoblotting using antibodies against Vif-HA and A3G-V5 or A3F-V5.

    Techniques Used: Transfection, Plasmid Preparation, Mutagenesis, Immunoprecipitation

    Identification of two regions in HIV-1 Vif that are required for the interaction with CBF-β. (A) Interaction of Vif mutants in the 84 GxSIEW 89 region with cellular factors. HEK293T cells were transfected with the vector control, WT or a Vif mutant. Cells were harvested 48 h later and subjected to immunoprecipitation analysis using the anti-HA antibody conjugated to agarose beads. Co-precipitated proteins were analyzed by immunoblotting with antibodies against Vif-HA, CUL5, CBF-β and ELOB. (C) Interaction of Vif mutants in the L 102 ADQLI 107 region with cellular factors. HEK293T cells were transfected with the vector control, WT or a Vif mutant. Cells were harvested 48 h later and subjected to immunoprecipitation analysis using the anti-HA antibody conjugated to agarose beads. Co-precipitated proteins were analyzed by immunoblotting using antibodies against Vif-HA, CUL5, CBF-β and ELOB. (B and D) Relative binding capacity of WT Vif (100%) and Vif mutants to CBF-β or CUL5. Error bars indicate the standard deviation from triplicate experiments.
    Figure Legend Snippet: Identification of two regions in HIV-1 Vif that are required for the interaction with CBF-β. (A) Interaction of Vif mutants in the 84 GxSIEW 89 region with cellular factors. HEK293T cells were transfected with the vector control, WT or a Vif mutant. Cells were harvested 48 h later and subjected to immunoprecipitation analysis using the anti-HA antibody conjugated to agarose beads. Co-precipitated proteins were analyzed by immunoblotting with antibodies against Vif-HA, CUL5, CBF-β and ELOB. (C) Interaction of Vif mutants in the L 102 ADQLI 107 region with cellular factors. HEK293T cells were transfected with the vector control, WT or a Vif mutant. Cells were harvested 48 h later and subjected to immunoprecipitation analysis using the anti-HA antibody conjugated to agarose beads. Co-precipitated proteins were analyzed by immunoblotting using antibodies against Vif-HA, CUL5, CBF-β and ELOB. (B and D) Relative binding capacity of WT Vif (100%) and Vif mutants to CBF-β or CUL5. Error bars indicate the standard deviation from triplicate experiments.

    Techniques Used: Transfection, Plasmid Preparation, Mutagenesis, Immunoprecipitation, Binding Assay, Standard Deviation

    4) Product Images from "Conserved and non-conserved features of HIV-1 and SIVagm Vif mediated suppression of APOBEC3 cytidine deaminases"

    Article Title: Conserved and non-conserved features of HIV-1 and SIVagm Vif mediated suppression of APOBEC3 cytidine deaminases

    Journal: Cellular microbiology

    doi: 10.1111/j.1462-5822.2008.01157.x

    Interaction of SIVagm Vif with A3C. A. 293T cells were co-transfected with an expression vector for SIVagmTan Vif plus an expression vector for HA-tagged A3A, A3C, A3F or A3G at a 2:1 ratio. Cells were harvested 48 h later and subjected to immunoprecipitation analysis using the anti-HA antibody conjugated to agarose beads. Cell lysates or coprecipitated proteins were analysed by immunoblotting using an anti-HA antibody to detect HA-tagged APOBEC3 proteins and an anti-myc antibody to detect myc-tagged SIVagmTan Vif. B. Co-precipitation of HIV-1 Vif-myc or SIVagmVif-myc with A3C-HA, as determined by co-precipitation analysis.
    Figure Legend Snippet: Interaction of SIVagm Vif with A3C. A. 293T cells were co-transfected with an expression vector for SIVagmTan Vif plus an expression vector for HA-tagged A3A, A3C, A3F or A3G at a 2:1 ratio. Cells were harvested 48 h later and subjected to immunoprecipitation analysis using the anti-HA antibody conjugated to agarose beads. Cell lysates or coprecipitated proteins were analysed by immunoblotting using an anti-HA antibody to detect HA-tagged APOBEC3 proteins and an anti-myc antibody to detect myc-tagged SIVagmTan Vif. B. Co-precipitation of HIV-1 Vif-myc or SIVagmVif-myc with A3C-HA, as determined by co-precipitation analysis.

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Immunoprecipitation

    Regions of A3C that are necessary for Vif-induced degradation. A. The chimeras produced between A3A and A3C were generated in regions between the predicted secondary structures of A3A and A3C. A3CΔN contains an in-frame deletion of amino acids 2-47. B. Various expression vectors for parental A3C-HA or chimeras were co-transfected with SIVagmTan Vif-myc or a control vector into 293T cells at a 1:3 ratio of APOBEC and SIVagmTan Vif-myc. Transfected cells were harvested 48 h after transfection and analysed by immunoblotting using anti-HA antibody to detect A3C-HA, anti-myc antibody to detect myc-tagged SIVagmTan Vif and an antibody against ribosomal P19 to assess protein loading. C. Expression vectors for A3C-HA or A3CΔN-HA (in-frame deletion of amino acids 2-47) were co-transfected with SIVagmTanVif-myc or SIVagmTanVifC117S-myc expression vector into 293T cells. The ratio of APOBEC and Vif is 1:3. Cells were harvested 48 h after transfection and analysed by immunoblotting using anti-HA antibody to detect A3C-HA or A3CΔN-HA, anti-myc antibody to detect myc-tagged SIVagmTanVif-myc or SIVagmTanVifC117S-myc and an antibody against ribosomal P19 to assess protein loading. D. 293T cells were co-transfected with an expression vector for SIVagmTan Vif plus various expression vectors for parental A3C-HA or chimeras at a 2:1 ratio. Cells were harvested 48 h later and subjected to immunoprecipitation analysis using the anti-HA antibody conjugated to agarose beads. Cell lysates or coprecipitated proteins were analysed by immunoblotting using an anti-HA antibody to detect HA-tagged A3C or chemeras proteins and an anti-myc antibody to detect myc-tagged SIVagmTan Vif.
    Figure Legend Snippet: Regions of A3C that are necessary for Vif-induced degradation. A. The chimeras produced between A3A and A3C were generated in regions between the predicted secondary structures of A3A and A3C. A3CΔN contains an in-frame deletion of amino acids 2-47. B. Various expression vectors for parental A3C-HA or chimeras were co-transfected with SIVagmTan Vif-myc or a control vector into 293T cells at a 1:3 ratio of APOBEC and SIVagmTan Vif-myc. Transfected cells were harvested 48 h after transfection and analysed by immunoblotting using anti-HA antibody to detect A3C-HA, anti-myc antibody to detect myc-tagged SIVagmTan Vif and an antibody against ribosomal P19 to assess protein loading. C. Expression vectors for A3C-HA or A3CΔN-HA (in-frame deletion of amino acids 2-47) were co-transfected with SIVagmTanVif-myc or SIVagmTanVifC117S-myc expression vector into 293T cells. The ratio of APOBEC and Vif is 1:3. Cells were harvested 48 h after transfection and analysed by immunoblotting using anti-HA antibody to detect A3C-HA or A3CΔN-HA, anti-myc antibody to detect myc-tagged SIVagmTanVif-myc or SIVagmTanVifC117S-myc and an antibody against ribosomal P19 to assess protein loading. D. 293T cells were co-transfected with an expression vector for SIVagmTan Vif plus various expression vectors for parental A3C-HA or chimeras at a 2:1 ratio. Cells were harvested 48 h later and subjected to immunoprecipitation analysis using the anti-HA antibody conjugated to agarose beads. Cell lysates or coprecipitated proteins were analysed by immunoblotting using an anti-HA antibody to detect HA-tagged A3C or chemeras proteins and an anti-myc antibody to detect myc-tagged SIVagmTan Vif.

    Techniques Used: Produced, Generated, Expressing, Transfection, Plasmid Preparation, Immunoprecipitation

    5) Product Images from "Integrator complex regulates NELF-mediated RNA polymerase II pause/release and processivity at coding genes"

    Article Title: Integrator complex regulates NELF-mediated RNA polymerase II pause/release and processivity at coding genes

    Journal: Nature Communications

    doi: 10.1038/ncomms6531

    Immuno-purification of NELF. ( a ) Flag/HA-epitope-tagged NELF-E (eNELF-E) from HeLa S3 Dignam nuclear extracts was sequentially immunopurified on anti-Flag and anti-HA antibody-conjugated agarose beads. Purified material was separated by SDS–PAGE and visualised by silver staining. eNELF-E-associated proteins were identified by MS (see Supplementary Dataset 1 ). ( b ) Flag/HA IPs from samples shown in ( a ) were separated by SDS–PAGE and the presence of eNELF-E-associated proteins identified was confirmed by immunoblotting. ( c ) Glycerol gradient sedimentation analysis of eNELF-E. Flag-purified eNELF-E-associated complexes were separated by centrifugation through a 12–40% glycerol gradient. Material of even-numbered fractions was resolved by SDS–PAGE and probed for identified proteins. ( d ) Reciprocal IPs (ReIPs): Flag-purified eNELF-E (Input) was subjected to IP using anti-Spt5, anti-INTS13, anti-HA antibodies or irrelevant rabbit IgG (IPr) or mouse IgG (IPm). Input, IP, as well as flow through (FT) were probed for eNELF-associated proteins.
    Figure Legend Snippet: Immuno-purification of NELF. ( a ) Flag/HA-epitope-tagged NELF-E (eNELF-E) from HeLa S3 Dignam nuclear extracts was sequentially immunopurified on anti-Flag and anti-HA antibody-conjugated agarose beads. Purified material was separated by SDS–PAGE and visualised by silver staining. eNELF-E-associated proteins were identified by MS (see Supplementary Dataset 1 ). ( b ) Flag/HA IPs from samples shown in ( a ) were separated by SDS–PAGE and the presence of eNELF-E-associated proteins identified was confirmed by immunoblotting. ( c ) Glycerol gradient sedimentation analysis of eNELF-E. Flag-purified eNELF-E-associated complexes were separated by centrifugation through a 12–40% glycerol gradient. Material of even-numbered fractions was resolved by SDS–PAGE and probed for identified proteins. ( d ) Reciprocal IPs (ReIPs): Flag-purified eNELF-E (Input) was subjected to IP using anti-Spt5, anti-INTS13, anti-HA antibodies or irrelevant rabbit IgG (IPr) or mouse IgG (IPm). Input, IP, as well as flow through (FT) were probed for eNELF-associated proteins.

    Techniques Used: Purification, SDS Page, Silver Staining, Mass Spectrometry, Sedimentation, Centrifugation, Flow Cytometry

    6) Product Images from "Requirement of HIV-1 Vif C-terminus for Vif-CBF-β interaction and assembly of CUL5-containing E3 ligase"

    Article Title: Requirement of HIV-1 Vif C-terminus for Vif-CBF-β interaction and assembly of CUL5-containing E3 ligase

    Journal: BMC Microbiology

    doi: 10.1186/s12866-014-0290-7

    Certain residues in HCCH region of Vif are important for interaction with CBF-β and/or CUL5. (A) Illustration of Vif mutant constructs. (B) . Amino acids 124-141 located in HCCH region of Vif involved in CBF-β/CUL5 interaction. HEK293T cells were transfected with VR1012 as a control vector or WT or various Vif mutants as indicated. Cells were harvested 48 h later and subjected to immunoprecipitation analysis using the anti-HA antibody conjugated to agarose beads. Co-precipitated proteins were analyzed by Western blotting against Vif-HA, CUL5, CBF-β and ELoB. (C) Relative binding capacity of WT Vif (100%) and Vif mutants to CBF-β or CUL5. Error bars represent the standard deviation from triplicate experiments.
    Figure Legend Snippet: Certain residues in HCCH region of Vif are important for interaction with CBF-β and/or CUL5. (A) Illustration of Vif mutant constructs. (B) . Amino acids 124-141 located in HCCH region of Vif involved in CBF-β/CUL5 interaction. HEK293T cells were transfected with VR1012 as a control vector or WT or various Vif mutants as indicated. Cells were harvested 48 h later and subjected to immunoprecipitation analysis using the anti-HA antibody conjugated to agarose beads. Co-precipitated proteins were analyzed by Western blotting against Vif-HA, CUL5, CBF-β and ELoB. (C) Relative binding capacity of WT Vif (100%) and Vif mutants to CBF-β or CUL5. Error bars represent the standard deviation from triplicate experiments.

    Techniques Used: Mutagenesis, Construct, Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Binding Assay, Standard Deviation

    The HCCH motif in primate lentiviral Vif proteins is involved in CBF-β/CUL5 interactions. HCCH or SLQ-AAA mutants in HIV-1 Vif (A) , SIVtan Vif (B) or SIVmac Vif (C) were transfected into HEK293T cells as indicated. Cells were harvested 48 h later and subjected to immunoprecipitation analysis using the anti-HA antibody conjugated to agarose beads. Co-precipitated proteins were analyzed by Western blotting against Vif-HA, CUL5, CBF-β and ELOB.
    Figure Legend Snippet: The HCCH motif in primate lentiviral Vif proteins is involved in CBF-β/CUL5 interactions. HCCH or SLQ-AAA mutants in HIV-1 Vif (A) , SIVtan Vif (B) or SIVmac Vif (C) were transfected into HEK293T cells as indicated. Cells were harvested 48 h later and subjected to immunoprecipitation analysis using the anti-HA antibody conjugated to agarose beads. Co-precipitated proteins were analyzed by Western blotting against Vif-HA, CUL5, CBF-β and ELOB.

    Techniques Used: Transfection, Immunoprecipitation, Western Blot

    Effects of C-terminal truncations on HIV-1 Vif function. (A) Construction of C-terminal truncated Vif mutants with an N-terminal HA tag. (B) Interactions of various truncated Vif mutants with cellular factors. HEK293T cells were transfected with VR1012 as a control vector or WT or truncated Vif mutants as indicated. Cells were harvested 48 h later and subjected to immunoprecipitation analysis using the anti-HA antibody conjugated to agarose beads. Co-precipitated proteins were analyzed by Western blotting against Vif-HA, CUL5, CBF-β and ELOB. (C) Effects of WT and truncated Vif proteins on A3G degradation and virion packaging. HEK293T cells were co-transfected with NL4-3ΔVif and A3G along with VR1012 as a control vector or WT or various truncated Vifs as indicated. A3G expression was assessed by Western blotting against A3G-HA, Vif-HA and tubulin as loading control. A3G packaging was evaluated by Western blotting against A3G-HA and CAp24 after virus was purified from the supernatant of cell cultures. (D) Effects of WT and truncated Vif proteins on antiviral activity of A3G. HIV-1 viruses were produced as described for panel C. Virus infectivity was assessed using MAGI indicator cells, with the virus infectivity in the presence of WT Vif set to 100%. Error bars represent the standard deviation from triplicate wells.
    Figure Legend Snippet: Effects of C-terminal truncations on HIV-1 Vif function. (A) Construction of C-terminal truncated Vif mutants with an N-terminal HA tag. (B) Interactions of various truncated Vif mutants with cellular factors. HEK293T cells were transfected with VR1012 as a control vector or WT or truncated Vif mutants as indicated. Cells were harvested 48 h later and subjected to immunoprecipitation analysis using the anti-HA antibody conjugated to agarose beads. Co-precipitated proteins were analyzed by Western blotting against Vif-HA, CUL5, CBF-β and ELOB. (C) Effects of WT and truncated Vif proteins on A3G degradation and virion packaging. HEK293T cells were co-transfected with NL4-3ΔVif and A3G along with VR1012 as a control vector or WT or various truncated Vifs as indicated. A3G expression was assessed by Western blotting against A3G-HA, Vif-HA and tubulin as loading control. A3G packaging was evaluated by Western blotting against A3G-HA and CAp24 after virus was purified from the supernatant of cell cultures. (D) Effects of WT and truncated Vif proteins on antiviral activity of A3G. HIV-1 viruses were produced as described for panel C. Virus infectivity was assessed using MAGI indicator cells, with the virus infectivity in the presence of WT Vif set to 100%. Error bars represent the standard deviation from triplicate wells.

    Techniques Used: Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Expressing, Purification, Activity Assay, Produced, Infection, Standard Deviation

    Unaltered interactions of Vif mutants with A3G or A3F imply no changes in conformation from WT Vif protein HEK293T cells were transfected with the vector control or WT or Vif mutant plus A3G-V5 (A) or A3F-V5 (B). Cells were treated with 10 μM MG132 12 h prior to harvesting, and then Vif-HA was immunoprecipitated from cell lysates with the anti-HA antibody conjugated to agarose beads. The interaction of Vif-HA with A3G-V5 or A3F-V5 was detected by Western blotting against Vif-HA and A3G-V5 or A3F-V5.
    Figure Legend Snippet: Unaltered interactions of Vif mutants with A3G or A3F imply no changes in conformation from WT Vif protein HEK293T cells were transfected with the vector control or WT or Vif mutant plus A3G-V5 (A) or A3F-V5 (B). Cells were treated with 10 μM MG132 12 h prior to harvesting, and then Vif-HA was immunoprecipitated from cell lysates with the anti-HA antibody conjugated to agarose beads. The interaction of Vif-HA with A3G-V5 or A3F-V5 was detected by Western blotting against Vif-HA and A3G-V5 or A3F-V5.

    Techniques Used: Transfection, Plasmid Preparation, Mutagenesis, Immunoprecipitation, Western Blot

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    Lysis:

    Article Title: Distinct Determinants in HIV-1 Vif and Human APOBEC3 Proteins Are Required for the Suppression of Diverse Host Anti-Viral Proteins
    Article Snippet: Immunoprecipitation and immunoblot analysis A T-25 flask of HEK293T cells was transfected with 3 µg APOBEC3 plus 3 µg of wild-type or mutant Vif expression vectors as indicated. .. Cells were harvested, washed twice with cold PBS and lysed in lysis buffer (50 mM Tris–HCl [pH 7.5] with 150 mM NaCl, 1% [v/v] Triton X-100, and complete protease inhibitor cocktail tablets) at 4°C for 1 h, then centrifuged at 10,000g for 30 min. For myc-tag immunoprecipitation, pre-cleared cell lysates were mixed with anti-myc antibody (Upstate) and incubated with protein G beads at 4°C for 3 h. For HA tag immunoprecipitation, pre-cleared cell lysates were mixed with anti-HA antibody-conjugated agarose beads (Roche) and incubated at 4°C for 3 h. Samples were then washed three times with washing buffer (20 mM Tris–HCl [pH 7.5], with 100 mM NaCl, 0.1 mM EDTA, and 0.05% [v/v] Tween-20). ..

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    Protease Inhibitor:

    Article Title: Distinct Determinants in HIV-1 Vif and Human APOBEC3 Proteins Are Required for the Suppression of Diverse Host Anti-Viral Proteins
    Article Snippet: Immunoprecipitation and immunoblot analysis A T-25 flask of HEK293T cells was transfected with 3 µg APOBEC3 plus 3 µg of wild-type or mutant Vif expression vectors as indicated. .. Cells were harvested, washed twice with cold PBS and lysed in lysis buffer (50 mM Tris–HCl [pH 7.5] with 150 mM NaCl, 1% [v/v] Triton X-100, and complete protease inhibitor cocktail tablets) at 4°C for 1 h, then centrifuged at 10,000g for 30 min. For myc-tag immunoprecipitation, pre-cleared cell lysates were mixed with anti-myc antibody (Upstate) and incubated with protein G beads at 4°C for 3 h. For HA tag immunoprecipitation, pre-cleared cell lysates were mixed with anti-HA antibody-conjugated agarose beads (Roche) and incubated at 4°C for 3 h. Samples were then washed three times with washing buffer (20 mM Tris–HCl [pH 7.5], with 100 mM NaCl, 0.1 mM EDTA, and 0.05% [v/v] Tween-20). ..

    Article Title: Self-Association of Gata1 Enhances Transcriptional Activity In Vivo in Zebra Fish Embryos
    Article Snippet: Anti-HA antibody was purchased from Roche Diagnostics. .. For analyzing Gata1-Fog1 interaction, MBP-Gata1 fusion proteins were mixed with FLAG-tagged Fog1 protein in a binding buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM mercaptoethanol, 1× protease inhibitor cocktail [Roche Diagnostics], 10 μM ZnSO4 ) and incubated with amylose resin for 1 h. For analyzing Gata1 self-association, HA-tagged Gata1 proteins were mixed with FLAG-tagged proteins in a binding buffer (33 mM Tris-HCl [pH 7.5], 100 mM NaCl, 3.3 mM EDTA, 0.67 mM dithiothreitol, 0.33% NP-40, 1× protease inhibitor cocktail) and incubated with anti-HA antibody-conjugated agarose beads (Roche Diagnostics) for 4 h. The beads were collected by centrifugation at 13,000 × g for 1 min and washed three times in binding buffer. .. Precipitated proteins were eluted in SDS sample buffer and resolved on SDS-10% polyacrylamide gel electrophoresis, followed by immunoblot analysis with anti-MBP (Santa Cruz), anti-HA (Roche Diagnostics), and anti-FLAG (Sigma) antibodies.

    Article Title: Inhibition of Vpx-Mediated SAMHD1 and Vpr-Mediated Host Helicase Transcription Factor Degradation by Selective Disruption of Viral CRL4 (DCAF1) E3 Ubiquitin Ligase Assembly
    Article Snippet: DNA transfection was carried out using Lipofectamine 2000 (Invitrogen; catalog no. 52887) according to the manufacturer's instructions. .. For coimmunoprecipitation assays, HEK 293T cells were harvested at 48 h after transfection, washed twice with cold phosphate-buffered saline (PBS), lysed in lysis buffer (150 mM Tris, pH 7.5, with 150 mM NaCl, 1% Triton X-100, and complete protease inhibitor cocktail tablets [(Roche]) at 4°C for 30 min, and then centrifuged for clarification at 10,000 × g for 30 min at 4°C. .. Precleared cell lysates were mixed with anti-HA antibody-conjugated agarose beads (Roche; catalog no. 190-119) or anti-Flag antibody-conjugated agarose beads (Sigma; A-2220) and incubated at 4°C for 3 h or overnight.

    Immunoprecipitation:

    Article Title: Distinct Determinants in HIV-1 Vif and Human APOBEC3 Proteins Are Required for the Suppression of Diverse Host Anti-Viral Proteins
    Article Snippet: Immunoprecipitation and immunoblot analysis A T-25 flask of HEK293T cells was transfected with 3 µg APOBEC3 plus 3 µg of wild-type or mutant Vif expression vectors as indicated. .. Cells were harvested, washed twice with cold PBS and lysed in lysis buffer (50 mM Tris–HCl [pH 7.5] with 150 mM NaCl, 1% [v/v] Triton X-100, and complete protease inhibitor cocktail tablets) at 4°C for 1 h, then centrifuged at 10,000g for 30 min. For myc-tag immunoprecipitation, pre-cleared cell lysates were mixed with anti-myc antibody (Upstate) and incubated with protein G beads at 4°C for 3 h. For HA tag immunoprecipitation, pre-cleared cell lysates were mixed with anti-HA antibody-conjugated agarose beads (Roche) and incubated at 4°C for 3 h. Samples were then washed three times with washing buffer (20 mM Tris–HCl [pH 7.5], with 100 mM NaCl, 0.1 mM EDTA, and 0.05% [v/v] Tween-20). ..

    Incubation:

    Article Title: Distinct Determinants in HIV-1 Vif and Human APOBEC3 Proteins Are Required for the Suppression of Diverse Host Anti-Viral Proteins
    Article Snippet: Immunoprecipitation and immunoblot analysis A T-25 flask of HEK293T cells was transfected with 3 µg APOBEC3 plus 3 µg of wild-type or mutant Vif expression vectors as indicated. .. Cells were harvested, washed twice with cold PBS and lysed in lysis buffer (50 mM Tris–HCl [pH 7.5] with 150 mM NaCl, 1% [v/v] Triton X-100, and complete protease inhibitor cocktail tablets) at 4°C for 1 h, then centrifuged at 10,000g for 30 min. For myc-tag immunoprecipitation, pre-cleared cell lysates were mixed with anti-myc antibody (Upstate) and incubated with protein G beads at 4°C for 3 h. For HA tag immunoprecipitation, pre-cleared cell lysates were mixed with anti-HA antibody-conjugated agarose beads (Roche) and incubated at 4°C for 3 h. Samples were then washed three times with washing buffer (20 mM Tris–HCl [pH 7.5], with 100 mM NaCl, 0.1 mM EDTA, and 0.05% [v/v] Tween-20). ..

    Article Title: Self-Association of Gata1 Enhances Transcriptional Activity In Vivo in Zebra Fish Embryos
    Article Snippet: Anti-HA antibody was purchased from Roche Diagnostics. .. For analyzing Gata1-Fog1 interaction, MBP-Gata1 fusion proteins were mixed with FLAG-tagged Fog1 protein in a binding buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM mercaptoethanol, 1× protease inhibitor cocktail [Roche Diagnostics], 10 μM ZnSO4 ) and incubated with amylose resin for 1 h. For analyzing Gata1 self-association, HA-tagged Gata1 proteins were mixed with FLAG-tagged proteins in a binding buffer (33 mM Tris-HCl [pH 7.5], 100 mM NaCl, 3.3 mM EDTA, 0.67 mM dithiothreitol, 0.33% NP-40, 1× protease inhibitor cocktail) and incubated with anti-HA antibody-conjugated agarose beads (Roche Diagnostics) for 4 h. The beads were collected by centrifugation at 13,000 × g for 1 min and washed three times in binding buffer. .. Precipitated proteins were eluted in SDS sample buffer and resolved on SDS-10% polyacrylamide gel electrophoresis, followed by immunoblot analysis with anti-MBP (Santa Cruz), anti-HA (Roche Diagnostics), and anti-FLAG (Sigma) antibodies.

    Binding Assay:

    Article Title: Self-Association of Gata1 Enhances Transcriptional Activity In Vivo in Zebra Fish Embryos
    Article Snippet: Anti-HA antibody was purchased from Roche Diagnostics. .. For analyzing Gata1-Fog1 interaction, MBP-Gata1 fusion proteins were mixed with FLAG-tagged Fog1 protein in a binding buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM mercaptoethanol, 1× protease inhibitor cocktail [Roche Diagnostics], 10 μM ZnSO4 ) and incubated with amylose resin for 1 h. For analyzing Gata1 self-association, HA-tagged Gata1 proteins were mixed with FLAG-tagged proteins in a binding buffer (33 mM Tris-HCl [pH 7.5], 100 mM NaCl, 3.3 mM EDTA, 0.67 mM dithiothreitol, 0.33% NP-40, 1× protease inhibitor cocktail) and incubated with anti-HA antibody-conjugated agarose beads (Roche Diagnostics) for 4 h. The beads were collected by centrifugation at 13,000 × g for 1 min and washed three times in binding buffer. .. Precipitated proteins were eluted in SDS sample buffer and resolved on SDS-10% polyacrylamide gel electrophoresis, followed by immunoblot analysis with anti-MBP (Santa Cruz), anti-HA (Roche Diagnostics), and anti-FLAG (Sigma) antibodies.

    Centrifugation:

    Article Title: Self-Association of Gata1 Enhances Transcriptional Activity In Vivo in Zebra Fish Embryos
    Article Snippet: Anti-HA antibody was purchased from Roche Diagnostics. .. For analyzing Gata1-Fog1 interaction, MBP-Gata1 fusion proteins were mixed with FLAG-tagged Fog1 protein in a binding buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM mercaptoethanol, 1× protease inhibitor cocktail [Roche Diagnostics], 10 μM ZnSO4 ) and incubated with amylose resin for 1 h. For analyzing Gata1 self-association, HA-tagged Gata1 proteins were mixed with FLAG-tagged proteins in a binding buffer (33 mM Tris-HCl [pH 7.5], 100 mM NaCl, 3.3 mM EDTA, 0.67 mM dithiothreitol, 0.33% NP-40, 1× protease inhibitor cocktail) and incubated with anti-HA antibody-conjugated agarose beads (Roche Diagnostics) for 4 h. The beads were collected by centrifugation at 13,000 × g for 1 min and washed three times in binding buffer. .. Precipitated proteins were eluted in SDS sample buffer and resolved on SDS-10% polyacrylamide gel electrophoresis, followed by immunoblot analysis with anti-MBP (Santa Cruz), anti-HA (Roche Diagnostics), and anti-FLAG (Sigma) antibodies.

    Transfection:

    Article Title: Inhibition of Vpx-Mediated SAMHD1 and Vpr-Mediated Host Helicase Transcription Factor Degradation by Selective Disruption of Viral CRL4 (DCAF1) E3 Ubiquitin Ligase Assembly
    Article Snippet: DNA transfection was carried out using Lipofectamine 2000 (Invitrogen; catalog no. 52887) according to the manufacturer's instructions. .. For coimmunoprecipitation assays, HEK 293T cells were harvested at 48 h after transfection, washed twice with cold phosphate-buffered saline (PBS), lysed in lysis buffer (150 mM Tris, pH 7.5, with 150 mM NaCl, 1% Triton X-100, and complete protease inhibitor cocktail tablets [(Roche]) at 4°C for 30 min, and then centrifuged for clarification at 10,000 × g for 30 min at 4°C. .. Precleared cell lysates were mixed with anti-HA antibody-conjugated agarose beads (Roche; catalog no. 190-119) or anti-Flag antibody-conjugated agarose beads (Sigma; A-2220) and incubated at 4°C for 3 h or overnight.

    Clarification Assay:

    Article Title: Inhibition of Vpx-Mediated SAMHD1 and Vpr-Mediated Host Helicase Transcription Factor Degradation by Selective Disruption of Viral CRL4 (DCAF1) E3 Ubiquitin Ligase Assembly
    Article Snippet: DNA transfection was carried out using Lipofectamine 2000 (Invitrogen; catalog no. 52887) according to the manufacturer's instructions. .. For coimmunoprecipitation assays, HEK 293T cells were harvested at 48 h after transfection, washed twice with cold phosphate-buffered saline (PBS), lysed in lysis buffer (150 mM Tris, pH 7.5, with 150 mM NaCl, 1% Triton X-100, and complete protease inhibitor cocktail tablets [(Roche]) at 4°C for 30 min, and then centrifuged for clarification at 10,000 × g for 30 min at 4°C. .. Precleared cell lysates were mixed with anti-HA antibody-conjugated agarose beads (Roche; catalog no. 190-119) or anti-Flag antibody-conjugated agarose beads (Sigma; A-2220) and incubated at 4°C for 3 h or overnight.

    Affinity Purification:

    Article Title: MEP50/PRMT5-mediated methylation activates GLI1 in Hedgehog signalling through inhibition of ubiquitination by the ITCH/NUMB complex
    Article Snippet: The supernatants were immunoprecipitated with anti-FLAG antibody-conjugated M2 agarose (400 μl; Sigma-Aldrich). .. Bound proteins eluted 0.4 mg ml−1 FLAG peptide (Sigma-Aldrich) were further affinity purified using 20 μl anti-HA antibody-conjugated agarose (Roche Diagnostics). .. The final elutes from HA beads eluted with 2.5 mg ml−1 HA peptide (Roche Diagnostics) were separated by SDS–PAGE on a 5–20% gradient gel for silver staining analysis.

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    Roche anti ha antibody conjugated agarose beads
    The D 14 RMR 17 and Trp79 domains mediate the interaction between Vif and A3DE. HIV-1 Vif DR14/15 and W79 all showed reduced interaction with A3DE when compared to WT Vif. HEK293T cells were cotransfected with A3DE and a control vector, WT Vif, or one of the indicated Vif mutants. At 48 h post-transfection, cell lysates were prepared and immunoprecipitated with <t>anti-myc</t> <t>antibody</t> and <t>agarose-conjugated</t> protein A/G. Cell lysates (A) and the interaction of A3C with WT or mutant Vif molecules(B) were detected by immunoblotting with antibodies against <t>A3DE-HA</t> and Vif-myc.
    Anti Ha Antibody Conjugated Agarose Beads, supplied by Roche, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti ha antibody conjugated agarose beads/product/Roche
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti ha antibody conjugated agarose beads - by Bioz Stars, 2021-06
    86/100 stars
      Buy from Supplier

    86
    Roche anti ha ab conjugated agarose beads
    BIV Vif recruits Cul2-EloB-EloC and MVV Vif recruits Cul5-EloB-EloC to induce btA3Z2-Z3 and oaA3Z2-Z3 degradation. 293 T cells (5 × 10 6 ) were transfected with 10 μg <t>HA-tagged</t> BIV Vif or 10 μg VHL and 10 μg VR1012 as a positive and a negative control, respectively (A, B) ; or with 10 μg HA-tagged MVV Vif or 10 μg HIV Vif and 10 μg VR1012 as a positive and a negative control, respectively (C, D) . After 48 h of transfection, cell lysates were immunoprecipitated with HA <t>beads,</t> followed by SDS-PAGE and immunoblot analysis using <t>anti-HA,</t> <t>anti-hCul2,</t> anti-hCul5, anti-hEloB, anti-hEloC and anti-hCBF-β <t>antibodies.</t> All immunoprecipitation experiments were repeated three times.
    Anti Ha Ab Conjugated Agarose Beads, supplied by Roche, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti ha ab conjugated agarose beads/product/Roche
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti ha ab conjugated agarose beads - by Bioz Stars, 2021-06
    86/100 stars
      Buy from Supplier

    Image Search Results


    The D 14 RMR 17 and Trp79 domains mediate the interaction between Vif and A3DE. HIV-1 Vif DR14/15 and W79 all showed reduced interaction with A3DE when compared to WT Vif. HEK293T cells were cotransfected with A3DE and a control vector, WT Vif, or one of the indicated Vif mutants. At 48 h post-transfection, cell lysates were prepared and immunoprecipitated with anti-myc antibody and agarose-conjugated protein A/G. Cell lysates (A) and the interaction of A3C with WT or mutant Vif molecules(B) were detected by immunoblotting with antibodies against A3DE-HA and Vif-myc.

    Journal: PLoS ONE

    Article Title: Distinct Determinants in HIV-1 Vif and Human APOBEC3 Proteins Are Required for the Suppression of Diverse Host Anti-Viral Proteins

    doi: 10.1371/journal.pone.0003963

    Figure Lengend Snippet: The D 14 RMR 17 and Trp79 domains mediate the interaction between Vif and A3DE. HIV-1 Vif DR14/15 and W79 all showed reduced interaction with A3DE when compared to WT Vif. HEK293T cells were cotransfected with A3DE and a control vector, WT Vif, or one of the indicated Vif mutants. At 48 h post-transfection, cell lysates were prepared and immunoprecipitated with anti-myc antibody and agarose-conjugated protein A/G. Cell lysates (A) and the interaction of A3C with WT or mutant Vif molecules(B) were detected by immunoblotting with antibodies against A3DE-HA and Vif-myc.

    Article Snippet: Cells were harvested, washed twice with cold PBS and lysed in lysis buffer (50 mM Tris–HCl [pH 7.5] with 150 mM NaCl, 1% [v/v] Triton X-100, and complete protease inhibitor cocktail tablets) at 4°C for 1 h, then centrifuged at 10,000g for 30 min. For myc-tag immunoprecipitation, pre-cleared cell lysates were mixed with anti-myc antibody (Upstate) and incubated with protein G beads at 4°C for 3 h. For HA tag immunoprecipitation, pre-cleared cell lysates were mixed with anti-HA antibody-conjugated agarose beads (Roche) and incubated at 4°C for 3 h. Samples were then washed three times with washing buffer (20 mM Tris–HCl [pH 7.5], with 100 mM NaCl, 0.1 mM EDTA, and 0.05% [v/v] Tween-20).

    Techniques: Plasmid Preparation, Transfection, Immunoprecipitation, Mutagenesis

    The C-CCD of A3F behaves like the full-length A3F. (A) Alignment of A3C and the C-CCD of AF. (B) Alignment of A3C and the N-CCD of AF. (C) Interaction of A3F and A3F-C with HIV-1 Vif. HEK293T cells were cotransfected with HIV-1 Vif-myc plus control vector, full-length A3F-HA, or A3F-C-HA. The cells were treated with 10 µM MG132 12 h prior to harvesting., and A3F-HA proteins were immunoprecipitated from cell lysates with an anti-HA antibody conjugated to agarose beads. The interaction of A3F with HIV-1 Vif molecules was detected by immunoblotting with antibodies against A3F-HA and Vif antibody. (D) HIV-1 Vif induces the degradation of the C-CCD of A3F. HEK293T cells were transfected with an expression vector encoding the C-CCD of A3F plus a control vector, WT Vif, or one of the indicated Vif mutant expression vectors. A3F-C stability was assessed by immunoblotting with antibodies against V5, Vif-myc, and β-tubulin as a loading control.

    Journal: PLoS ONE

    Article Title: Distinct Determinants in HIV-1 Vif and Human APOBEC3 Proteins Are Required for the Suppression of Diverse Host Anti-Viral Proteins

    doi: 10.1371/journal.pone.0003963

    Figure Lengend Snippet: The C-CCD of A3F behaves like the full-length A3F. (A) Alignment of A3C and the C-CCD of AF. (B) Alignment of A3C and the N-CCD of AF. (C) Interaction of A3F and A3F-C with HIV-1 Vif. HEK293T cells were cotransfected with HIV-1 Vif-myc plus control vector, full-length A3F-HA, or A3F-C-HA. The cells were treated with 10 µM MG132 12 h prior to harvesting., and A3F-HA proteins were immunoprecipitated from cell lysates with an anti-HA antibody conjugated to agarose beads. The interaction of A3F with HIV-1 Vif molecules was detected by immunoblotting with antibodies against A3F-HA and Vif antibody. (D) HIV-1 Vif induces the degradation of the C-CCD of A3F. HEK293T cells were transfected with an expression vector encoding the C-CCD of A3F plus a control vector, WT Vif, or one of the indicated Vif mutant expression vectors. A3F-C stability was assessed by immunoblotting with antibodies against V5, Vif-myc, and β-tubulin as a loading control.

    Article Snippet: Cells were harvested, washed twice with cold PBS and lysed in lysis buffer (50 mM Tris–HCl [pH 7.5] with 150 mM NaCl, 1% [v/v] Triton X-100, and complete protease inhibitor cocktail tablets) at 4°C for 1 h, then centrifuged at 10,000g for 30 min. For myc-tag immunoprecipitation, pre-cleared cell lysates were mixed with anti-myc antibody (Upstate) and incubated with protein G beads at 4°C for 3 h. For HA tag immunoprecipitation, pre-cleared cell lysates were mixed with anti-HA antibody-conjugated agarose beads (Roche) and incubated at 4°C for 3 h. Samples were then washed three times with washing buffer (20 mM Tris–HCl [pH 7.5], with 100 mM NaCl, 0.1 mM EDTA, and 0.05% [v/v] Tween-20).

    Techniques: Plasmid Preparation, Immunoprecipitation, Transfection, Expressing, Mutagenesis

    The D 14 RMR 17 and Trp79 domains mediate the interaction between HIV-1 Vif and A3C. Vif DR14/15 and W79 showed reduced interaction with A3C when compared to WT Vif. HEK293T cells were cotransfected with A3C and the control vector, HIV-1 Vif, or one of the indicated Vif mutants. At 48 h post-transfection, cell lysates were prepared and immunoprecipitated with anti-myc antibody and agarose-conjugated protein A/G. Cell lysate (A) and the interaction of A3C with WT or mutant Vif molecules(B) were detected by immunoblotting with antibodies against A3G-HA and Vif-myc.

    Journal: PLoS ONE

    Article Title: Distinct Determinants in HIV-1 Vif and Human APOBEC3 Proteins Are Required for the Suppression of Diverse Host Anti-Viral Proteins

    doi: 10.1371/journal.pone.0003963

    Figure Lengend Snippet: The D 14 RMR 17 and Trp79 domains mediate the interaction between HIV-1 Vif and A3C. Vif DR14/15 and W79 showed reduced interaction with A3C when compared to WT Vif. HEK293T cells were cotransfected with A3C and the control vector, HIV-1 Vif, or one of the indicated Vif mutants. At 48 h post-transfection, cell lysates were prepared and immunoprecipitated with anti-myc antibody and agarose-conjugated protein A/G. Cell lysate (A) and the interaction of A3C with WT or mutant Vif molecules(B) were detected by immunoblotting with antibodies against A3G-HA and Vif-myc.

    Article Snippet: Cells were harvested, washed twice with cold PBS and lysed in lysis buffer (50 mM Tris–HCl [pH 7.5] with 150 mM NaCl, 1% [v/v] Triton X-100, and complete protease inhibitor cocktail tablets) at 4°C for 1 h, then centrifuged at 10,000g for 30 min. For myc-tag immunoprecipitation, pre-cleared cell lysates were mixed with anti-myc antibody (Upstate) and incubated with protein G beads at 4°C for 3 h. For HA tag immunoprecipitation, pre-cleared cell lysates were mixed with anti-HA antibody-conjugated agarose beads (Roche) and incubated at 4°C for 3 h. Samples were then washed three times with washing buffer (20 mM Tris–HCl [pH 7.5], with 100 mM NaCl, 0.1 mM EDTA, and 0.05% [v/v] Tween-20).

    Techniques: Plasmid Preparation, Transfection, Immunoprecipitation, Mutagenesis

    Self-association of zebra fish Gata1 proteins. HA-tagged (lanes 1 and 2) and FLAG (FL)-tagged (lanes 3 and 4) proteins of wild-type Gata1 (WT) (lanes 1 and 3) and Gata1KA6 (lanes 2 and 4) were examined by immunoblot (IB) analysis with anti-FLAG (αFL) (upper panel) or anti-HA (lower panel) antibodies. Arrows indicate migration positions of the Gata1 proteins. Mixtures of FLAG-tagged and HA-tagged proteins (lanes 5 and 8), FLAG-tagged protein alone (lanes 6 and 9), HA-tagged protein alone (lanes 7 and 10) for wild-type Gata1 (lanes 5 to 7) or Gata1KA6 (lanes 8 to 10) were immunoprecipitated with anti-HA-conjugated agarose beads. Precipitated proteins were analyzed by immunoblotting.

    Journal: Molecular and Cellular Biology

    Article Title: Self-Association of Gata1 Enhances Transcriptional Activity In Vivo in Zebra Fish Embryos

    doi: 10.1128/MCB.23.22.8295-8305.2003

    Figure Lengend Snippet: Self-association of zebra fish Gata1 proteins. HA-tagged (lanes 1 and 2) and FLAG (FL)-tagged (lanes 3 and 4) proteins of wild-type Gata1 (WT) (lanes 1 and 3) and Gata1KA6 (lanes 2 and 4) were examined by immunoblot (IB) analysis with anti-FLAG (αFL) (upper panel) or anti-HA (lower panel) antibodies. Arrows indicate migration positions of the Gata1 proteins. Mixtures of FLAG-tagged and HA-tagged proteins (lanes 5 and 8), FLAG-tagged protein alone (lanes 6 and 9), HA-tagged protein alone (lanes 7 and 10) for wild-type Gata1 (lanes 5 to 7) or Gata1KA6 (lanes 8 to 10) were immunoprecipitated with anti-HA-conjugated agarose beads. Precipitated proteins were analyzed by immunoblotting.

    Article Snippet: For analyzing Gata1-Fog1 interaction, MBP-Gata1 fusion proteins were mixed with FLAG-tagged Fog1 protein in a binding buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM mercaptoethanol, 1× protease inhibitor cocktail [Roche Diagnostics], 10 μM ZnSO4 ) and incubated with amylose resin for 1 h. For analyzing Gata1 self-association, HA-tagged Gata1 proteins were mixed with FLAG-tagged proteins in a binding buffer (33 mM Tris-HCl [pH 7.5], 100 mM NaCl, 3.3 mM EDTA, 0.67 mM dithiothreitol, 0.33% NP-40, 1× protease inhibitor cocktail) and incubated with anti-HA antibody-conjugated agarose beads (Roche Diagnostics) for 4 h. The beads were collected by centrifugation at 13,000 × g for 1 min and washed three times in binding buffer.

    Techniques: Fluorescence In Situ Hybridization, Migration, Immunoprecipitation

    Vif mutants maintain interactions with A3G and A3F. HEK293T cells were transfected with the vector control, WT or a Vif mutant plus A3G-V5 (A) or A3F-V5 (B). Cells were treated with 10 µM MG132 12 h prior to harvesting, and then Vif-HA was immunoprecipitated from cell lysates with an anti-HA antibody conjugated to agarose beads. The interaction of Vif-HA with A3G-V5 or A3F-V5 was detected by immunoblotting using antibodies against Vif-HA and A3G-V5 or A3F-V5.

    Journal: PLoS ONE

    Article Title: Identification of HIV-1 Vif Regions Required for CBF-? Interaction and APOBEC3 Suppression

    doi: 10.1371/journal.pone.0095738

    Figure Lengend Snippet: Vif mutants maintain interactions with A3G and A3F. HEK293T cells were transfected with the vector control, WT or a Vif mutant plus A3G-V5 (A) or A3F-V5 (B). Cells were treated with 10 µM MG132 12 h prior to harvesting, and then Vif-HA was immunoprecipitated from cell lysates with an anti-HA antibody conjugated to agarose beads. The interaction of Vif-HA with A3G-V5 or A3F-V5 was detected by immunoblotting using antibodies against Vif-HA and A3G-V5 or A3F-V5.

    Article Snippet: For HA-tag immunoprecipitation, pre-cleared cell lysates were mixed with anti-HA antibody-conjugated agarose beads (Roche) and incubated at 4°C for 3 h. Samples were then washed six times with washing buffer (20 mM Tris-HCl [pH 7.5], with 100 mM NaCl, 0.1 mM EDTA and 0.05% [v/v] Tween-20).

    Techniques: Transfection, Plasmid Preparation, Mutagenesis, Immunoprecipitation

    Identification of two regions in HIV-1 Vif that are required for the interaction with CBF-β. (A) Interaction of Vif mutants in the 84 GxSIEW 89 region with cellular factors. HEK293T cells were transfected with the vector control, WT or a Vif mutant. Cells were harvested 48 h later and subjected to immunoprecipitation analysis using the anti-HA antibody conjugated to agarose beads. Co-precipitated proteins were analyzed by immunoblotting with antibodies against Vif-HA, CUL5, CBF-β and ELOB. (C) Interaction of Vif mutants in the L 102 ADQLI 107 region with cellular factors. HEK293T cells were transfected with the vector control, WT or a Vif mutant. Cells were harvested 48 h later and subjected to immunoprecipitation analysis using the anti-HA antibody conjugated to agarose beads. Co-precipitated proteins were analyzed by immunoblotting using antibodies against Vif-HA, CUL5, CBF-β and ELOB. (B and D) Relative binding capacity of WT Vif (100%) and Vif mutants to CBF-β or CUL5. Error bars indicate the standard deviation from triplicate experiments.

    Journal: PLoS ONE

    Article Title: Identification of HIV-1 Vif Regions Required for CBF-? Interaction and APOBEC3 Suppression

    doi: 10.1371/journal.pone.0095738

    Figure Lengend Snippet: Identification of two regions in HIV-1 Vif that are required for the interaction with CBF-β. (A) Interaction of Vif mutants in the 84 GxSIEW 89 region with cellular factors. HEK293T cells were transfected with the vector control, WT or a Vif mutant. Cells were harvested 48 h later and subjected to immunoprecipitation analysis using the anti-HA antibody conjugated to agarose beads. Co-precipitated proteins were analyzed by immunoblotting with antibodies against Vif-HA, CUL5, CBF-β and ELOB. (C) Interaction of Vif mutants in the L 102 ADQLI 107 region with cellular factors. HEK293T cells were transfected with the vector control, WT or a Vif mutant. Cells were harvested 48 h later and subjected to immunoprecipitation analysis using the anti-HA antibody conjugated to agarose beads. Co-precipitated proteins were analyzed by immunoblotting using antibodies against Vif-HA, CUL5, CBF-β and ELOB. (B and D) Relative binding capacity of WT Vif (100%) and Vif mutants to CBF-β or CUL5. Error bars indicate the standard deviation from triplicate experiments.

    Article Snippet: For HA-tag immunoprecipitation, pre-cleared cell lysates were mixed with anti-HA antibody-conjugated agarose beads (Roche) and incubated at 4°C for 3 h. Samples were then washed six times with washing buffer (20 mM Tris-HCl [pH 7.5], with 100 mM NaCl, 0.1 mM EDTA and 0.05% [v/v] Tween-20).

    Techniques: Transfection, Plasmid Preparation, Mutagenesis, Immunoprecipitation, Binding Assay, Standard Deviation

    BIV Vif recruits Cul2-EloB-EloC and MVV Vif recruits Cul5-EloB-EloC to induce btA3Z2-Z3 and oaA3Z2-Z3 degradation. 293 T cells (5 × 10 6 ) were transfected with 10 μg HA-tagged BIV Vif or 10 μg VHL and 10 μg VR1012 as a positive and a negative control, respectively (A, B) ; or with 10 μg HA-tagged MVV Vif or 10 μg HIV Vif and 10 μg VR1012 as a positive and a negative control, respectively (C, D) . After 48 h of transfection, cell lysates were immunoprecipitated with HA beads, followed by SDS-PAGE and immunoblot analysis using anti-HA, anti-hCul2, anti-hCul5, anti-hEloB, anti-hEloC and anti-hCBF-β antibodies. All immunoprecipitation experiments were repeated three times.

    Journal: Retrovirology

    Article Title: Role of cullin-elonginB-elonginC E3 complex in bovine immunodeficiency virus and maedi-visna virus Vif-mediated degradation of host A3Z2-Z3 proteins

    doi: 10.1186/s12977-014-0077-9

    Figure Lengend Snippet: BIV Vif recruits Cul2-EloB-EloC and MVV Vif recruits Cul5-EloB-EloC to induce btA3Z2-Z3 and oaA3Z2-Z3 degradation. 293 T cells (5 × 10 6 ) were transfected with 10 μg HA-tagged BIV Vif or 10 μg VHL and 10 μg VR1012 as a positive and a negative control, respectively (A, B) ; or with 10 μg HA-tagged MVV Vif or 10 μg HIV Vif and 10 μg VR1012 as a positive and a negative control, respectively (C, D) . After 48 h of transfection, cell lysates were immunoprecipitated with HA beads, followed by SDS-PAGE and immunoblot analysis using anti-HA, anti-hCul2, anti-hCul5, anti-hEloB, anti-hEloC and anti-hCBF-β antibodies. All immunoprecipitation experiments were repeated three times.

    Article Snippet: The pre-cleared supernatants were collected and then mix with anti-HA Ab-conjugated agarose beads (Roche, Mannheim, Germany), followed by incubation at 4°C for 3 h. Alternatively, the pre-cleared supernatants were collected and incubated with mouse anti-cmyc (Millipore) for 1 h and then mix with Protein G-agarose (Roche), followed by incubation at 4°C for 3 h. The beads were washed three times with wash buffer (20 mM Tris, pH 7.5, with 100 mM NaCl, 0.1 mM EDTA and 0.05% Tween 20), and the pellet was resuspended in 30 μl glycine HCl (pH 2.0) elution buffer.

    Techniques: Transfection, Negative Control, Immunoprecipitation, SDS Page

    BIV Vif interacts with btCul2 and MVV Vif with oaCul5, while CBF-β are not involved. (A–C) 293 T cells (5 × 10 6 ) were co-transfected with 5 μg cmyc-tagged BIV Vif (MVV Vif or HIV Vif) and 5 μg VR1012, 5 μg Flag-tagged btCBF-β (oaCBF-β or hCBF-β) and 5 μg VR1012, 5 μg cmyc-tagged BIV Vif (MVV Vif or HIV Vif) and 5 μg Flag-tagged btCBF-β (oaCBF-β or hCBF-β). At 48 h after transfection, cell lysates were co-immunoprecipitated with an anti-cmyc antibody, followed by SDS-PAGE and immunoblot analysis using anti-cmyc and anti-Flag antibodies. (D-F) 293 T cells (5 × 10 6 ) were co-transfected with 6 μg HA-tagged BIV Vif (MVV Vif, or pVHL) and 10 μg VR1012, 10 μg cmyc-tagged btCul2 (oaCul2 or hCul2) and 6 μg VR1012, 6 μg HA-tagged BIV Vif (MVV Vif or pVHL) and 10 μg cmyc-tagged btCul2 (oaCul2 or hCul2). Cells were co-immunoprecipitated with HA beads followed by SDS-PAGE and immunoblot analysis using anti-HA and anti-cmyc antibodies. (G-I) 293 T cells (5 × 10 6 ) were co-transfected with 4 μg HA-tagged BIV Vif (MVV Vif or HIV Vif) and 8 μg VR1012, 8 μg cmyc-tagged btCul5 (oaCul5 or hCul5) and 4 μg VR1012, 4 μg HA-tagged BIV Vif (MVV Vif or HIV Vif) and 8 μg cmyc-tagged btCul5 (oaCul5 or hCul5). Cells were co-immunoprecipitated with HA beads followed by SDS-PAGE and immunoblot analysis using an anti-HA and anti-cmyc antibodies. All co-immunoprecipitation experiments were repeated three times.

    Journal: Retrovirology

    Article Title: Role of cullin-elonginB-elonginC E3 complex in bovine immunodeficiency virus and maedi-visna virus Vif-mediated degradation of host A3Z2-Z3 proteins

    doi: 10.1186/s12977-014-0077-9

    Figure Lengend Snippet: BIV Vif interacts with btCul2 and MVV Vif with oaCul5, while CBF-β are not involved. (A–C) 293 T cells (5 × 10 6 ) were co-transfected with 5 μg cmyc-tagged BIV Vif (MVV Vif or HIV Vif) and 5 μg VR1012, 5 μg Flag-tagged btCBF-β (oaCBF-β or hCBF-β) and 5 μg VR1012, 5 μg cmyc-tagged BIV Vif (MVV Vif or HIV Vif) and 5 μg Flag-tagged btCBF-β (oaCBF-β or hCBF-β). At 48 h after transfection, cell lysates were co-immunoprecipitated with an anti-cmyc antibody, followed by SDS-PAGE and immunoblot analysis using anti-cmyc and anti-Flag antibodies. (D-F) 293 T cells (5 × 10 6 ) were co-transfected with 6 μg HA-tagged BIV Vif (MVV Vif, or pVHL) and 10 μg VR1012, 10 μg cmyc-tagged btCul2 (oaCul2 or hCul2) and 6 μg VR1012, 6 μg HA-tagged BIV Vif (MVV Vif or pVHL) and 10 μg cmyc-tagged btCul2 (oaCul2 or hCul2). Cells were co-immunoprecipitated with HA beads followed by SDS-PAGE and immunoblot analysis using anti-HA and anti-cmyc antibodies. (G-I) 293 T cells (5 × 10 6 ) were co-transfected with 4 μg HA-tagged BIV Vif (MVV Vif or HIV Vif) and 8 μg VR1012, 8 μg cmyc-tagged btCul5 (oaCul5 or hCul5) and 4 μg VR1012, 4 μg HA-tagged BIV Vif (MVV Vif or HIV Vif) and 8 μg cmyc-tagged btCul5 (oaCul5 or hCul5). Cells were co-immunoprecipitated with HA beads followed by SDS-PAGE and immunoblot analysis using an anti-HA and anti-cmyc antibodies. All co-immunoprecipitation experiments were repeated three times.

    Article Snippet: The pre-cleared supernatants were collected and then mix with anti-HA Ab-conjugated agarose beads (Roche, Mannheim, Germany), followed by incubation at 4°C for 3 h. Alternatively, the pre-cleared supernatants were collected and incubated with mouse anti-cmyc (Millipore) for 1 h and then mix with Protein G-agarose (Roche), followed by incubation at 4°C for 3 h. The beads were washed three times with wash buffer (20 mM Tris, pH 7.5, with 100 mM NaCl, 0.1 mM EDTA and 0.05% Tween 20), and the pellet was resuspended in 30 μl glycine HCl (pH 2.0) elution buffer.

    Techniques: Transfection, Immunoprecipitation, SDS Page

    The C-x1-C-x1-H-x19-C motif is crucial and predicted to be a zinc binding loop. (A) Alignment of partial BIV Vif sequences with primate lentiviral Vifs by BioEdit. (B) 293 T cells (0.5 × 10 6 ) were co-transfected with 30 ng HA-tagged btA3Z2-Z3 and 200 ng cmyc-tagged BIV Vif or BIV Vif mutants H102L, C111S, C113S, H115L, C134S, H149L or C111S/C113S. At 48 h after transfection, the cells were harvested for Western blotting using anti-HA, anti-cmyc and anti-tubulin antibodies. ( C and D ) 293 T cells (1 × 10 6 ) were co-transfected with 1 μg pNL4-3ΔVif plus 15 ng VR1012, or HA-tagged btA3Z2-Z3 and 100 ng cmyc-tagged BIV Vif, BIV Vif C111S/C113S or VR1012. The virus infectivity was assayed by the MAGI assay. Virus infectivity was set to 100% in the absence of btA3Z2-Z3. (D) Western blot was performed on the cell lysates from (C) to show the producer cell levels of btA3Z2-Z3 protein (anti-HA), BIV Vif/BIV Vif C111S/C113 (anti-cmyc) and tubulin. (E) 293 T cells (5 × 10 6 ) were co-transfected with 10 μg cmyc-tagged btCul2 and 6 μg HA-tagged BIV Vif or 6 μg BIV Vif C111S/C113S. At 48 h after transfection, cells were immunoprecipitated with HA beads, followed by SDS-PAGE and immunoblot analysis using an anti-HA antibody and an anti-cmyc antibody. (F and G) Panels A and B are views of the alpha carbon ribbon and differ from each other by 90 degrees. Residues which likely participate in the coordination of zinc ions are shown and labeled. All infection experiments were repeated three times.

    Journal: Retrovirology

    Article Title: Role of cullin-elonginB-elonginC E3 complex in bovine immunodeficiency virus and maedi-visna virus Vif-mediated degradation of host A3Z2-Z3 proteins

    doi: 10.1186/s12977-014-0077-9

    Figure Lengend Snippet: The C-x1-C-x1-H-x19-C motif is crucial and predicted to be a zinc binding loop. (A) Alignment of partial BIV Vif sequences with primate lentiviral Vifs by BioEdit. (B) 293 T cells (0.5 × 10 6 ) were co-transfected with 30 ng HA-tagged btA3Z2-Z3 and 200 ng cmyc-tagged BIV Vif or BIV Vif mutants H102L, C111S, C113S, H115L, C134S, H149L or C111S/C113S. At 48 h after transfection, the cells were harvested for Western blotting using anti-HA, anti-cmyc and anti-tubulin antibodies. ( C and D ) 293 T cells (1 × 10 6 ) were co-transfected with 1 μg pNL4-3ΔVif plus 15 ng VR1012, or HA-tagged btA3Z2-Z3 and 100 ng cmyc-tagged BIV Vif, BIV Vif C111S/C113S or VR1012. The virus infectivity was assayed by the MAGI assay. Virus infectivity was set to 100% in the absence of btA3Z2-Z3. (D) Western blot was performed on the cell lysates from (C) to show the producer cell levels of btA3Z2-Z3 protein (anti-HA), BIV Vif/BIV Vif C111S/C113 (anti-cmyc) and tubulin. (E) 293 T cells (5 × 10 6 ) were co-transfected with 10 μg cmyc-tagged btCul2 and 6 μg HA-tagged BIV Vif or 6 μg BIV Vif C111S/C113S. At 48 h after transfection, cells were immunoprecipitated with HA beads, followed by SDS-PAGE and immunoblot analysis using an anti-HA antibody and an anti-cmyc antibody. (F and G) Panels A and B are views of the alpha carbon ribbon and differ from each other by 90 degrees. Residues which likely participate in the coordination of zinc ions are shown and labeled. All infection experiments were repeated three times.

    Article Snippet: The pre-cleared supernatants were collected and then mix with anti-HA Ab-conjugated agarose beads (Roche, Mannheim, Germany), followed by incubation at 4°C for 3 h. Alternatively, the pre-cleared supernatants were collected and incubated with mouse anti-cmyc (Millipore) for 1 h and then mix with Protein G-agarose (Roche), followed by incubation at 4°C for 3 h. The beads were washed three times with wash buffer (20 mM Tris, pH 7.5, with 100 mM NaCl, 0.1 mM EDTA and 0.05% Tween 20), and the pellet was resuspended in 30 μl glycine HCl (pH 2.0) elution buffer.

    Techniques: Binding Assay, Transfection, Western Blot, Infection, Immunoprecipitation, SDS Page, Labeling