Structured Review

Addgene inc pcdna3 dn hcul5 flag

Pcdna3 Dn Hcul5 Flag, supplied by Addgene inc, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/pcdna3 dn hcul5 flag/product/Addgene inc
Average 91 stars, based on 1 article reviews
Price from $9.99 to $1999.99
pcdna3 dn hcul5 flag - by Bioz Stars, 2023-02
91/100 stars

Images

1) Product Images from "The Balance between Mono- and NEDD8-Chains Controlled by NEDP1 upon DNA Damage Is a Regulatory Module of the HSP70 ATPase Activity"

Article Title: The Balance between Mono- and NEDD8-Chains Controlled by NEDP1 upon DNA Damage Is a Regulatory Module of the HSP70 ATPase Activity

Journal: Cell Reports

doi: 10.1016/j.celrep.2019.08.070


Figure Legend Snippet:

Techniques Used: Western Blot, Recombinant, Protease Inhibitor, Labeling, Isolation, Cell Viability Assay, Caspase-Glo Assay, Plasmid Preparation, Software


Structured Review

Addgene inc pcdna3 dn hcul5 flag
CUL5 is required for FIV Vif-induced degradation of feline APOBEC3s. (A) FIV Vif interacts with CUL5 but not CUL2. myc-CUL5 or myc-CUL2 expression plasmids were cotransfected with the FIV Vif-V5 expression plasmid. Cell lysates were immunoprecipitated with anti-myc beads and then analyzed by immunoblotting with anti-V5 antibody for FIV Vif and anti-myc antibody for CUL2 and CUL5. (B and C) Dominant negative CUL5 (DN-CUL5), but not DN-CUL2, disrupts the degradation of feline A3s induced by FIV Vif. HEK293T cells were cotransfected with 300 ng expression plasmids for FcaA3Z2b-HA, FcaA3Z3-HA, or FcaA3Z2bZ3-HA and 700 ng of DN-CUL5–FLAG or DN-CUL2–FLAG with 30 ng of FIV Vif-V5. <t>pcDNA3.1</t> was used as a control plasmid to replace the FIV Vif or dominant negative CUL5/2 expression plasmids. Cells were analyzed by immunoblotting using anti-HA, anti-V5, anti-CUL5, anti-Flag, and antitubulin antibodies. (D) HEK293T cells were cotransfected with 50, 100, or 200 ng expression plasmids for FcaA3Z2b-HA and 700 ng of DN–CUL2-FLAG with 30 ng of FIV Vif-V5. Immunoblotting was performed as described above for panel B. (E) FIV Vif induces the degradation of FcaA3s in a proteasome-dependent manner. HEK293T cells were transfected with HsaA3G-HA or FcaZ2bZ3-HA and HIV-1 Vif-V5 or FIV Vif-V5 expression plasmids; pcDNA3.1 was used as an empty plasmid control. The transfected cells were treated with the proteasome inhibitor MG132 (2.5, 5, 7.5, or 10 μM) or DMSO as a control at 36 h posttransfection. Cells were harvested 12 h later (48 h after transfection) and then analyzed by immunoblotting with anti-HA, anti-V5, and antitubulin antibodies. The percentages of FcaA3 and HsaA3G were calculated relative to those in the absence of FIV Vif or HIV-1 Vif (set as 1.00).
Pcdna3 Dn Hcul5 Flag, supplied by Addgene inc, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/pcdna3 dn hcul5 flag/product/Addgene inc
Average 91 stars, based on 1 article reviews
Price from $9.99 to $1999.99
pcdna3 dn hcul5 flag - by Bioz Stars, 2023-02
91/100 stars

Images

1) Product Images from "Identification of a Conserved Interface of Human Immunodeficiency Virus Type 1 and Feline Immunodeficiency Virus Vifs with Cullin 5"

Article Title: Identification of a Conserved Interface of Human Immunodeficiency Virus Type 1 and Feline Immunodeficiency Virus Vifs with Cullin 5

Journal: Journal of Virology

doi: 10.1128/JVI.01697-17

CUL5 is required for FIV Vif-induced degradation of feline APOBEC3s. (A) FIV Vif interacts with CUL5 but not CUL2. myc-CUL5 or myc-CUL2 expression plasmids were cotransfected with the FIV Vif-V5 expression plasmid. Cell lysates were immunoprecipitated with anti-myc beads and then analyzed by immunoblotting with anti-V5 antibody for FIV Vif and anti-myc antibody for CUL2 and CUL5. (B and C) Dominant negative CUL5 (DN-CUL5), but not DN-CUL2, disrupts the degradation of feline A3s induced by FIV Vif. HEK293T cells were cotransfected with 300 ng expression plasmids for FcaA3Z2b-HA, FcaA3Z3-HA, or FcaA3Z2bZ3-HA and 700 ng of DN-CUL5–FLAG or DN-CUL2–FLAG with 30 ng of FIV Vif-V5. pcDNA3.1 was used as a control plasmid to replace the FIV Vif or dominant negative CUL5/2 expression plasmids. Cells were analyzed by immunoblotting using anti-HA, anti-V5, anti-CUL5, anti-Flag, and antitubulin antibodies. (D) HEK293T cells were cotransfected with 50, 100, or 200 ng expression plasmids for FcaA3Z2b-HA and 700 ng of DN–CUL2-FLAG with 30 ng of FIV Vif-V5. Immunoblotting was performed as described above for panel B. (E) FIV Vif induces the degradation of FcaA3s in a proteasome-dependent manner. HEK293T cells were transfected with HsaA3G-HA or FcaZ2bZ3-HA and HIV-1 Vif-V5 or FIV Vif-V5 expression plasmids; pcDNA3.1 was used as an empty plasmid control. The transfected cells were treated with the proteasome inhibitor MG132 (2.5, 5, 7.5, or 10 μM) or DMSO as a control at 36 h posttransfection. Cells were harvested 12 h later (48 h after transfection) and then analyzed by immunoblotting with anti-HA, anti-V5, and antitubulin antibodies. The percentages of FcaA3 and HsaA3G were calculated relative to those in the absence of FIV Vif or HIV-1 Vif (set as 1.00).
Figure Legend Snippet: CUL5 is required for FIV Vif-induced degradation of feline APOBEC3s. (A) FIV Vif interacts with CUL5 but not CUL2. myc-CUL5 or myc-CUL2 expression plasmids were cotransfected with the FIV Vif-V5 expression plasmid. Cell lysates were immunoprecipitated with anti-myc beads and then analyzed by immunoblotting with anti-V5 antibody for FIV Vif and anti-myc antibody for CUL2 and CUL5. (B and C) Dominant negative CUL5 (DN-CUL5), but not DN-CUL2, disrupts the degradation of feline A3s induced by FIV Vif. HEK293T cells were cotransfected with 300 ng expression plasmids for FcaA3Z2b-HA, FcaA3Z3-HA, or FcaA3Z2bZ3-HA and 700 ng of DN-CUL5–FLAG or DN-CUL2–FLAG with 30 ng of FIV Vif-V5. pcDNA3.1 was used as a control plasmid to replace the FIV Vif or dominant negative CUL5/2 expression plasmids. Cells were analyzed by immunoblotting using anti-HA, anti-V5, anti-CUL5, anti-Flag, and antitubulin antibodies. (D) HEK293T cells were cotransfected with 50, 100, or 200 ng expression plasmids for FcaA3Z2b-HA and 700 ng of DN–CUL2-FLAG with 30 ng of FIV Vif-V5. Immunoblotting was performed as described above for panel B. (E) FIV Vif induces the degradation of FcaA3s in a proteasome-dependent manner. HEK293T cells were transfected with HsaA3G-HA or FcaZ2bZ3-HA and HIV-1 Vif-V5 or FIV Vif-V5 expression plasmids; pcDNA3.1 was used as an empty plasmid control. The transfected cells were treated with the proteasome inhibitor MG132 (2.5, 5, 7.5, or 10 μM) or DMSO as a control at 36 h posttransfection. Cells were harvested 12 h later (48 h after transfection) and then analyzed by immunoblotting with anti-HA, anti-V5, and antitubulin antibodies. The percentages of FcaA3 and HsaA3G were calculated relative to those in the absence of FIV Vif or HIV-1 Vif (set as 1.00).

Techniques Used: Expressing, Plasmid Preparation, Immunoprecipitation, Western Blot, Dominant Negative Mutation, Transfection

Relevance of FIV Vif N-terminal residues for interaction with CUL5. (A) Schematic structure of FIV Vif. The numbers indicate the positions of amino acids mutated to alanines. The relative positions of the feline A3Z2 and A3Z3 interaction sites (Z2 box and Z3 box) and the Elongin B/C interaction site (BC box) are represented. NTD, N-terminal domain. (B) Coimmunoprecipitation of wild-type (WT) FIV Vif and mutants with CUL5. myc-CUL5 plasmids or the empty pcDNA3.1 plasmid was cotransfected with expression plasmids for wild-type FIV Vif-V5 or FIV Vif mutants. Immunoprecipitated (IP) complexes were analyzed by immunoblotting with anti-V5 for FIV Vif and anti-myc for CUL5. (C and D) HEK293T cells were transfected with expression plasmids for FcaZ2bZ3-HA, wild-type FIV Vif-V5, or the indicated FIV Vif mutants or the pcDNA3.1 empty plasmid (C) or with FIV Vif-V5, the indicated FIV Vif mutants, T7-ELOC, or HA-ELOB and the pcDNA3.1 empty plasmid (D). Cells were harvested 48 h after transfection, and proteins of cell lysates (input) and immunoprecipitated complexes were analyzed by using immunoblots stained with anti-V5 antibody for FIV Vif, anti-HA antibody for FcaZ2bZ3-HA and HA-Elongin B, and anti-T7 antibody for T7-Elongin C. (E) HEK293T cells were transfected with expression plasmids for FcaA3Z2bZ3-HA and wild-type FIV Vif-V5 or the indicated FIV Vif mutants or with the empty pcDNA3.1 plasmid. Cells were harvested and analyzed by immunoblotting with anti-HA, anti-V5, and antitubulin antibodies, respectively.
Figure Legend Snippet: Relevance of FIV Vif N-terminal residues for interaction with CUL5. (A) Schematic structure of FIV Vif. The numbers indicate the positions of amino acids mutated to alanines. The relative positions of the feline A3Z2 and A3Z3 interaction sites (Z2 box and Z3 box) and the Elongin B/C interaction site (BC box) are represented. NTD, N-terminal domain. (B) Coimmunoprecipitation of wild-type (WT) FIV Vif and mutants with CUL5. myc-CUL5 plasmids or the empty pcDNA3.1 plasmid was cotransfected with expression plasmids for wild-type FIV Vif-V5 or FIV Vif mutants. Immunoprecipitated (IP) complexes were analyzed by immunoblotting with anti-V5 for FIV Vif and anti-myc for CUL5. (C and D) HEK293T cells were transfected with expression plasmids for FcaZ2bZ3-HA, wild-type FIV Vif-V5, or the indicated FIV Vif mutants or the pcDNA3.1 empty plasmid (C) or with FIV Vif-V5, the indicated FIV Vif mutants, T7-ELOC, or HA-ELOB and the pcDNA3.1 empty plasmid (D). Cells were harvested 48 h after transfection, and proteins of cell lysates (input) and immunoprecipitated complexes were analyzed by using immunoblots stained with anti-V5 antibody for FIV Vif, anti-HA antibody for FcaZ2bZ3-HA and HA-Elongin B, and anti-T7 antibody for T7-Elongin C. (E) HEK293T cells were transfected with expression plasmids for FcaA3Z2bZ3-HA and wild-type FIV Vif-V5 or the indicated FIV Vif mutants or with the empty pcDNA3.1 plasmid. Cells were harvested and analyzed by immunoblotting with anti-HA, anti-V5, and antitubulin antibodies, respectively.

Techniques Used: Plasmid Preparation, Expressing, Immunoprecipitation, Western Blot, Transfection, Staining

Identification of determinants in the C terminus of FIV Vif that regulate binding to CUL5. (A, top) Sequence alignment of FIV Vifs from different FIV strains, including 34TF10, C36, TM2, PRR, Shizuoka, Oma (Pallas's cats), and lion subtype E. (Bottom) Sequence alignment of C-terminal residues of FIV Vif (clone 34TF10) and HIV-1 Vif (clone NL4-3). KCCC is a motif of FIV Vif that is similar to the HIV-1 Vif zinc interaction motif HCCH. The boxed region indicates a conserved hydrophobic motif. Nonsimilar residues are indicated in black with no background, conservative residues are indicated in blue with a cyan background, blocks of similar residues are indicated in black with a green background, identical residues are indicated in red with a yellow background, and weakly similar residues are indicated in green with no background. (B) Structure of the HIV-1 Vif-CUL5 complex (orange, Vif; green, CUL5) (PDB accession number 4N9F). A closeup view of the HIV-1 Vif-CUL5 interface is shown. The residues that are involved in the HIV-1 Vif-CUL5 interaction are indicated. Red dashed lines represent hydrogen bonds. (C) The FIV Vif 174IR175 region is important for the interaction of FIV Vif with CUL5. myc-CUL5 expression plasmids or the empty pcDNA3.1 plasmid was cotransfected with expression plasmids for wild-type FIV Vif-V5 or the indicated FIV Vif mutants. Immunoprecipitated complexes were analyzed by immunoblotting with anti-V5 for FIV Vif and with anti-myc for CUL5.
Figure Legend Snippet: Identification of determinants in the C terminus of FIV Vif that regulate binding to CUL5. (A, top) Sequence alignment of FIV Vifs from different FIV strains, including 34TF10, C36, TM2, PRR, Shizuoka, Oma (Pallas's cats), and lion subtype E. (Bottom) Sequence alignment of C-terminal residues of FIV Vif (clone 34TF10) and HIV-1 Vif (clone NL4-3). KCCC is a motif of FIV Vif that is similar to the HIV-1 Vif zinc interaction motif HCCH. The boxed region indicates a conserved hydrophobic motif. Nonsimilar residues are indicated in black with no background, conservative residues are indicated in blue with a cyan background, blocks of similar residues are indicated in black with a green background, identical residues are indicated in red with a yellow background, and weakly similar residues are indicated in green with no background. (B) Structure of the HIV-1 Vif-CUL5 complex (orange, Vif; green, CUL5) (PDB accession number 4N9F). A closeup view of the HIV-1 Vif-CUL5 interface is shown. The residues that are involved in the HIV-1 Vif-CUL5 interaction are indicated. Red dashed lines represent hydrogen bonds. (C) The FIV Vif 174IR175 region is important for the interaction of FIV Vif with CUL5. myc-CUL5 expression plasmids or the empty pcDNA3.1 plasmid was cotransfected with expression plasmids for wild-type FIV Vif-V5 or the indicated FIV Vif mutants. Immunoprecipitated complexes were analyzed by immunoblotting with anti-V5 for FIV Vif and with anti-myc for CUL5.

Techniques Used: Binding Assay, Sequencing, Expressing, Plasmid Preparation, Immunoprecipitation, Western Blot

Mutation of the 174IR175 region in FIV Vif does not impair interaction with FcaA3s, ELOB, and ELOC. (A) The FIV Vif 174175IR-AA mutant has lost degradation activity against FcaA3Z2bZ3. Cells were transfected with expression plasmids for FcaA3Z2bZ3-HA and wild-type FIV Vif-V5 or the indicated FIV Vif mutants or with the empty pcDNA3.1 plasmid. Cells were harvested and analyzed by immunoblotting with anti-HA, anti-V5, and antitubulin antibodies, respectively. (B) The FIV Vif 174175IR-AA mutant does not antagonize FcaA3Z2bZ3 antiviral activity. Single-round FIVΔvif luciferase reporter virions were produced in the presence of feline A3 expression plasmids (FcaA3Z2bZ3) with wild-type FIV Vif or Vif mutants; pcDNA3.1(+) was added as a control for feline A3 (control) and FIV Vif (vector). The infectivity of reporter vectors was determined by quantification of luciferase activity in HEK293T cells transduced with normalized amounts of viral vector particles. (C and D) The FIV Vif 174175IR-AA mutant still has the capability to bind to FcaA3s, ELOB, and ELOC. HEK293T cells were transfected with expression plasmids for FcaZ2bZ3-HA, wild-type FIV Vif-V5, or the indicated FIV Vif mutants or with the empty pcDNA3.1 plasmid (C) or with wild-type FIV Vif-V5, the indicated FIV Vif mutants, T7-ELOC, or HA-ELOB and the empty pcDNA3.1 plasmid (D). Cells were harvested 48 h after transfection, and proteins of cell lysates (input) and immunoprecipitated complexes were analyzed by using Western blots stained with anti-V5 antibody for FIV Vif, anti-HA antibody for FcaZ2bZ3-HA and HA-Elongin B, and anti-T7 antibody for T7-Elongin C. ***, P value of <0.001.
Figure Legend Snippet: Mutation of the 174IR175 region in FIV Vif does not impair interaction with FcaA3s, ELOB, and ELOC. (A) The FIV Vif 174175IR-AA mutant has lost degradation activity against FcaA3Z2bZ3. Cells were transfected with expression plasmids for FcaA3Z2bZ3-HA and wild-type FIV Vif-V5 or the indicated FIV Vif mutants or with the empty pcDNA3.1 plasmid. Cells were harvested and analyzed by immunoblotting with anti-HA, anti-V5, and antitubulin antibodies, respectively. (B) The FIV Vif 174175IR-AA mutant does not antagonize FcaA3Z2bZ3 antiviral activity. Single-round FIVΔvif luciferase reporter virions were produced in the presence of feline A3 expression plasmids (FcaA3Z2bZ3) with wild-type FIV Vif or Vif mutants; pcDNA3.1(+) was added as a control for feline A3 (control) and FIV Vif (vector). The infectivity of reporter vectors was determined by quantification of luciferase activity in HEK293T cells transduced with normalized amounts of viral vector particles. (C and D) The FIV Vif 174175IR-AA mutant still has the capability to bind to FcaA3s, ELOB, and ELOC. HEK293T cells were transfected with expression plasmids for FcaZ2bZ3-HA, wild-type FIV Vif-V5, or the indicated FIV Vif mutants or with the empty pcDNA3.1 plasmid (C) or with wild-type FIV Vif-V5, the indicated FIV Vif mutants, T7-ELOC, or HA-ELOB and the empty pcDNA3.1 plasmid (D). Cells were harvested 48 h after transfection, and proteins of cell lysates (input) and immunoprecipitated complexes were analyzed by using Western blots stained with anti-V5 antibody for FIV Vif, anti-HA antibody for FcaZ2bZ3-HA and HA-Elongin B, and anti-T7 antibody for T7-Elongin C. ***, P value of <0.001.

Techniques Used: Mutagenesis, Activity Assay, Transfection, Expressing, Plasmid Preparation, Western Blot, Luciferase, Produced, Infection, Transduction, Immunoprecipitation, Staining

FIV Vif-CUL5 three-dimensional structure model. (A) Homology model of the FIV Vif (orange)-CUL5 (green) complex in a cartoon representation. Helices α3 and α4 of Vif are interacting with CUL5. The model contains two unstructured loops (navy) before and after helix α2, as no structural template is available for these regions. These loops might be important for binding other parts of the complex. Residues that were subjected to mutational analysis are shown in a sphere representation. The region of the closeup shown in panel B is indicated by a light plum rectangle. (B) Closeup view of the homology model of the FIV Vif (orange)-CUL5 (green) complex in a cartoon representation, with interacting residues shown in a stick representation. (C) Expression plasmids for wild-type myc-CUL5 or the indicated mutants were cotransfected with the expression plasmid for wild-type FIV Vif-V5 into HEK293T cells. Cell lysates were immunoprecipitated with anti-myc beads and then analyzed by immunoblotting with anti-V5 antibody for FIV Vif and anti-myc antibody for CUL5. (D) HEK293T cells were cotransfected with expression plasmids for FcaA3Z2b-HA, FcaA3Z3-HA, or FcaA3Z2bZ3-HA and the CUL5 52LW53-AA mutant with FIV Vif-V5. pcDNA3.1 was used as a control plasmid to replace the FIV Vif or CUL5 expression plasmids. Cells were analyzed by immunoblotting using anti-HA, anti-V5, anti-CUL5, anti-Flag, and antitubulin antibodies. (E) Model of FIV Vif with the E3 ligase complex. The CUL5 interaction sites of FIV Vif (174IR175) are shown as red stars.
Figure Legend Snippet: FIV Vif-CUL5 three-dimensional structure model. (A) Homology model of the FIV Vif (orange)-CUL5 (green) complex in a cartoon representation. Helices α3 and α4 of Vif are interacting with CUL5. The model contains two unstructured loops (navy) before and after helix α2, as no structural template is available for these regions. These loops might be important for binding other parts of the complex. Residues that were subjected to mutational analysis are shown in a sphere representation. The region of the closeup shown in panel B is indicated by a light plum rectangle. (B) Closeup view of the homology model of the FIV Vif (orange)-CUL5 (green) complex in a cartoon representation, with interacting residues shown in a stick representation. (C) Expression plasmids for wild-type myc-CUL5 or the indicated mutants were cotransfected with the expression plasmid for wild-type FIV Vif-V5 into HEK293T cells. Cell lysates were immunoprecipitated with anti-myc beads and then analyzed by immunoblotting with anti-V5 antibody for FIV Vif and anti-myc antibody for CUL5. (D) HEK293T cells were cotransfected with expression plasmids for FcaA3Z2b-HA, FcaA3Z3-HA, or FcaA3Z2bZ3-HA and the CUL5 52LW53-AA mutant with FIV Vif-V5. pcDNA3.1 was used as a control plasmid to replace the FIV Vif or CUL5 expression plasmids. Cells were analyzed by immunoblotting using anti-HA, anti-V5, anti-CUL5, anti-Flag, and antitubulin antibodies. (E) Model of FIV Vif with the E3 ligase complex. The CUL5 interaction sites of FIV Vif (174IR175) are shown as red stars.

Techniques Used: Binding Assay, Expressing, Plasmid Preparation, Immunoprecipitation, Western Blot, Mutagenesis

FIV Vif binding to CUL5 is zinc independent. (A and B) HEK293T cells were transfected with expression plasmids for FcaZ2bZ3-HA (A) or HsaA3G-HA (B), and HIV-1 Vif-V5, or FIV Vif-V5. pcDNA3.1 was used as the empty plasmid control. Transfected cells were treated with the zinc chelator TPEN (2, 3, 4, 5, 6, 7, or 8 μM) or DMSO as a control at 36 h posttransfection. Cells were harvested 12 h later (48 h after transfection) and then analyzed by immunoblotting with anti-HA, anti-V5, and antitubulin antibodies. (C) The myc-CUL5 expression plasmid was cotransfected with FIV Vif-V5 or HIV-1 Vif-V5 expression plasmids into HEK293T cells. The transfected cells were treated with 5 μM TPEN or DMSO at 36 h posttransfection. Cell lysates were immunoprecipitated with anti-myc beads and then analyzed by immunoblotting with anti-V5 antibody for FIV Vif and HIV-1 Vif and with anti-myc antibody for CUL5.
Figure Legend Snippet: FIV Vif binding to CUL5 is zinc independent. (A and B) HEK293T cells were transfected with expression plasmids for FcaZ2bZ3-HA (A) or HsaA3G-HA (B), and HIV-1 Vif-V5, or FIV Vif-V5. pcDNA3.1 was used as the empty plasmid control. Transfected cells were treated with the zinc chelator TPEN (2, 3, 4, 5, 6, 7, or 8 μM) or DMSO as a control at 36 h posttransfection. Cells were harvested 12 h later (48 h after transfection) and then analyzed by immunoblotting with anti-HA, anti-V5, and antitubulin antibodies. (C) The myc-CUL5 expression plasmid was cotransfected with FIV Vif-V5 or HIV-1 Vif-V5 expression plasmids into HEK293T cells. The transfected cells were treated with 5 μM TPEN or DMSO at 36 h posttransfection. Cell lysates were immunoprecipitated with anti-myc beads and then analyzed by immunoblotting with anti-V5 antibody for FIV Vif and HIV-1 Vif and with anti-myc antibody for CUL5.

Techniques Used: Binding Assay, Transfection, Expressing, Plasmid Preparation, Western Blot, Immunoprecipitation

C184 of FIV Vif is essential for Vif-CUL5, Vif-FcaA3, and Vif-ELOB/C interactions. (A) Closeup view of the homology model of the FIV Vif (orange)-CUL5 (green) complex in a cartoon representation, with residues that underwent mutational analysis shown in a stick representation. These residues are K181, K182, C184, C187, and C192 in the loop following helix 3 of FIV Vif; C138 that forms a disulfide bond with C192 is also shown. (B) Sequence representation of the FIV Vif C-terminal domain (CTD). (C) myc-CUL5 or the empty pcDNA3.1 plasmid was cotransfected with expression plasmids for wild-type FIV Vif-V5 or the indicated FIV Vif mutants. Immunoprecipitated complexes were analyzed by immunoblotting with anti-V5 for FIV Vif and with anti-myc for CUL5. (D) HEK293T cells were transfected with expression plasmids for wild-type FIV Vif-V5 or the indicated FIV Vif mutants. Immunoprecipitated complexes were analyzed by immunoblotting with anti-V5 for FIV Vif and with anti-CUL5 for CUL5. (E and F) The FIV Vif C184S mutant has lost the capability to bind to FcaA3s, ELOB, and ELOC. HEK293T cells were transfected with expression plasmids for FcaZ2bZ3-HA, wild-type FIV Vif-V5, or the indicated FIV Vif mutants or with the empty pcDNA3.1 plasmid (E) or with wild-type FIV Vif-V5, the indicated FIV Vif mutants, T7-ELOC, or HA-ELOB and the empty pcDNA3.1 plasmid (F). Cells were harvested 48 h after transfection, and proteins of cell lysates (input) and immunoprecipitated complexes were analyzed by using Western blots stained with anti-V5 antibody for FIV Vif, anti-HA antibody for FcaZ2bZ3-HA and HA-Elongin B, and anti-T7 antibody for T7-Elongin C. (G) HEK293T cells were transfected with expression plasmids for FcaA3Z2bZ3-HA and wild-type FIV Vif-V5 or the indicated FIV Vif mutants or with the empty pcDNA3.1 plasmid. Cells were harvested and analyzed by immunoblotting with anti-HA, anti-V5, and antitubulin antibodies, respectively.
Figure Legend Snippet: C184 of FIV Vif is essential for Vif-CUL5, Vif-FcaA3, and Vif-ELOB/C interactions. (A) Closeup view of the homology model of the FIV Vif (orange)-CUL5 (green) complex in a cartoon representation, with residues that underwent mutational analysis shown in a stick representation. These residues are K181, K182, C184, C187, and C192 in the loop following helix 3 of FIV Vif; C138 that forms a disulfide bond with C192 is also shown. (B) Sequence representation of the FIV Vif C-terminal domain (CTD). (C) myc-CUL5 or the empty pcDNA3.1 plasmid was cotransfected with expression plasmids for wild-type FIV Vif-V5 or the indicated FIV Vif mutants. Immunoprecipitated complexes were analyzed by immunoblotting with anti-V5 for FIV Vif and with anti-myc for CUL5. (D) HEK293T cells were transfected with expression plasmids for wild-type FIV Vif-V5 or the indicated FIV Vif mutants. Immunoprecipitated complexes were analyzed by immunoblotting with anti-V5 for FIV Vif and with anti-CUL5 for CUL5. (E and F) The FIV Vif C184S mutant has lost the capability to bind to FcaA3s, ELOB, and ELOC. HEK293T cells were transfected with expression plasmids for FcaZ2bZ3-HA, wild-type FIV Vif-V5, or the indicated FIV Vif mutants or with the empty pcDNA3.1 plasmid (E) or with wild-type FIV Vif-V5, the indicated FIV Vif mutants, T7-ELOC, or HA-ELOB and the empty pcDNA3.1 plasmid (F). Cells were harvested 48 h after transfection, and proteins of cell lysates (input) and immunoprecipitated complexes were analyzed by using Western blots stained with anti-V5 antibody for FIV Vif, anti-HA antibody for FcaZ2bZ3-HA and HA-Elongin B, and anti-T7 antibody for T7-Elongin C. (G) HEK293T cells were transfected with expression plasmids for FcaA3Z2bZ3-HA and wild-type FIV Vif-V5 or the indicated FIV Vif mutants or with the empty pcDNA3.1 plasmid. Cells were harvested and analyzed by immunoblotting with anti-HA, anti-V5, and antitubulin antibodies, respectively.

Techniques Used: Sequencing, Plasmid Preparation, Expressing, Immunoprecipitation, Western Blot, Transfection, Mutagenesis, Staining

dominant negative dn cul5 expression plasmid pcdna3 dn hcul5 flag  (Addgene inc)

 
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 91

    Structured Review

    Addgene inc dominant negative dn cul5 expression plasmid pcdna3 dn hcul5 flag
    ( A ) Proteasomal degradation. 293T cells stably expressing HA-PPP2R5A were transfected with NL4-3 Vif in the presence of DMSO (control) or the proteasome inhibitor bortezomib and analysed by intracellular flow cytometry for HA. ( B ) <t>CUL5-dependent</t> degradation. 293T cells stably expressing GFP-PPP2R5B were co-transfected with NL4-3 Vif plus empty vector, wildtype <t>cullin-5</t> (CUL5 WT) or a dominant negative cullin-5 mutant (CUL5 DN) and analysed by flow cytometry for GFP. ( C ) CUL5 complex-dependent degradation. 293T cells stably expressing HA-PPP2R5B (upper panels) or HA-APOBEC3G (lower panels) were transduced with the indicated shRNA. Cells were then transfected with NL4-3 Vif and analysed by intracellular flow cytometry for HA. Green/red shading shows Vif-transfected cells in the indicated shRNA background. Red lines showing HA staining in cells transduced with control shRNA are included in each panel for reference. In all experiments, cells were analysed 36 hr post-transfection, and transfected cells determined by co-transfection with GFP ( A and C ) or mCherry ( B ). MFI values are shown for transfected (red/green) and untransfected (blue) cells. DOI: http://dx.doi.org/10.7554/eLife.18296.015
    Dominant Negative Dn Cul5 Expression Plasmid Pcdna3 Dn Hcul5 Flag, supplied by Addgene inc, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dominant negative dn cul5 expression plasmid pcdna3 dn hcul5 flag/product/Addgene inc
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    dominant negative dn cul5 expression plasmid pcdna3 dn hcul5 flag - by Bioz Stars, 2023-02
    91/100 stars

    Images

    1) Product Images from "Temporal proteomic analysis of HIV infection reveals remodelling of the host phosphoproteome by lentiviral Vif variants"

    Article Title: Temporal proteomic analysis of HIV infection reveals remodelling of the host phosphoproteome by lentiviral Vif variants

    Journal: eLife

    doi: 10.7554/eLife.18296

    ( A ) Proteasomal degradation. 293T cells stably expressing HA-PPP2R5A were transfected with NL4-3 Vif in the presence of DMSO (control) or the proteasome inhibitor bortezomib and analysed by intracellular flow cytometry for HA. ( B ) CUL5-dependent degradation. 293T cells stably expressing GFP-PPP2R5B were co-transfected with NL4-3 Vif plus empty vector, wildtype cullin-5 (CUL5 WT) or a dominant negative cullin-5 mutant (CUL5 DN) and analysed by flow cytometry for GFP. ( C ) CUL5 complex-dependent degradation. 293T cells stably expressing HA-PPP2R5B (upper panels) or HA-APOBEC3G (lower panels) were transduced with the indicated shRNA. Cells were then transfected with NL4-3 Vif and analysed by intracellular flow cytometry for HA. Green/red shading shows Vif-transfected cells in the indicated shRNA background. Red lines showing HA staining in cells transduced with control shRNA are included in each panel for reference. In all experiments, cells were analysed 36 hr post-transfection, and transfected cells determined by co-transfection with GFP ( A and C ) or mCherry ( B ). MFI values are shown for transfected (red/green) and untransfected (blue) cells. DOI: http://dx.doi.org/10.7554/eLife.18296.015
    Figure Legend Snippet: ( A ) Proteasomal degradation. 293T cells stably expressing HA-PPP2R5A were transfected with NL4-3 Vif in the presence of DMSO (control) or the proteasome inhibitor bortezomib and analysed by intracellular flow cytometry for HA. ( B ) CUL5-dependent degradation. 293T cells stably expressing GFP-PPP2R5B were co-transfected with NL4-3 Vif plus empty vector, wildtype cullin-5 (CUL5 WT) or a dominant negative cullin-5 mutant (CUL5 DN) and analysed by flow cytometry for GFP. ( C ) CUL5 complex-dependent degradation. 293T cells stably expressing HA-PPP2R5B (upper panels) or HA-APOBEC3G (lower panels) were transduced with the indicated shRNA. Cells were then transfected with NL4-3 Vif and analysed by intracellular flow cytometry for HA. Green/red shading shows Vif-transfected cells in the indicated shRNA background. Red lines showing HA staining in cells transduced with control shRNA are included in each panel for reference. In all experiments, cells were analysed 36 hr post-transfection, and transfected cells determined by co-transfection with GFP ( A and C ) or mCherry ( B ). MFI values are shown for transfected (red/green) and untransfected (blue) cells. DOI: http://dx.doi.org/10.7554/eLife.18296.015

    Techniques Used: Stable Transfection, Expressing, Transfection, Flow Cytometry, Plasmid Preparation, Dominant Negative Mutation, Mutagenesis, Transduction, shRNA, Staining, Cotransfection


    Structured Review

    Addgene inc pcdna3 dn hcul5 flag
    Pcdna3 Dn Hcul5 Flag, supplied by Addgene inc, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcdna3 dn hcul5 flag/product/Addgene inc
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pcdna3 dn hcul5 flag - by Bioz Stars, 2023-02
    91/100 stars

    Images

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 91
    Addgene inc pcdna3 dn hcul5 flag

    Pcdna3 Dn Hcul5 Flag, supplied by Addgene inc, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcdna3 dn hcul5 flag/product/Addgene inc
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pcdna3 dn hcul5 flag - by Bioz Stars, 2023-02
    91/100 stars
      Buy from Supplier

    91
    Addgene inc dominant negative dn cul5 expression plasmid pcdna3 dn hcul5 flag
    ( A ) Proteasomal degradation. 293T cells stably expressing HA-PPP2R5A were transfected with NL4-3 Vif in the presence of DMSO (control) or the proteasome inhibitor bortezomib and analysed by intracellular flow cytometry for HA. ( B ) <t>CUL5-dependent</t> degradation. 293T cells stably expressing GFP-PPP2R5B were co-transfected with NL4-3 Vif plus empty vector, wildtype <t>cullin-5</t> (CUL5 WT) or a dominant negative cullin-5 mutant (CUL5 DN) and analysed by flow cytometry for GFP. ( C ) CUL5 complex-dependent degradation. 293T cells stably expressing HA-PPP2R5B (upper panels) or HA-APOBEC3G (lower panels) were transduced with the indicated shRNA. Cells were then transfected with NL4-3 Vif and analysed by intracellular flow cytometry for HA. Green/red shading shows Vif-transfected cells in the indicated shRNA background. Red lines showing HA staining in cells transduced with control shRNA are included in each panel for reference. In all experiments, cells were analysed 36 hr post-transfection, and transfected cells determined by co-transfection with GFP ( A and C ) or mCherry ( B ). MFI values are shown for transfected (red/green) and untransfected (blue) cells. DOI: http://dx.doi.org/10.7554/eLife.18296.015
    Dominant Negative Dn Cul5 Expression Plasmid Pcdna3 Dn Hcul5 Flag, supplied by Addgene inc, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dominant negative dn cul5 expression plasmid pcdna3 dn hcul5 flag/product/Addgene inc
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    dominant negative dn cul5 expression plasmid pcdna3 dn hcul5 flag - by Bioz Stars, 2023-02
    91/100 stars
      Buy from Supplier

    Image Search Results


    Journal: Cell Reports

    Article Title: The Balance between Mono- and NEDD8-Chains Controlled by NEDP1 upon DNA Damage Is a Regulatory Module of the HSP70 ATPase Activity

    doi: 10.1016/j.celrep.2019.08.070

    Figure Lengend Snippet:

    Article Snippet: pcDNA3-DN-hCUL5-Flag , , Addgene plasmid #15823.

    Techniques: Western Blot, Recombinant, Protease Inhibitor, Labeling, Isolation, Cell Viability Assay, Caspase-Glo Assay, Plasmid Preparation, Software

    ( A ) Proteasomal degradation. 293T cells stably expressing HA-PPP2R5A were transfected with NL4-3 Vif in the presence of DMSO (control) or the proteasome inhibitor bortezomib and analysed by intracellular flow cytometry for HA. ( B ) CUL5-dependent degradation. 293T cells stably expressing GFP-PPP2R5B were co-transfected with NL4-3 Vif plus empty vector, wildtype cullin-5 (CUL5 WT) or a dominant negative cullin-5 mutant (CUL5 DN) and analysed by flow cytometry for GFP. ( C ) CUL5 complex-dependent degradation. 293T cells stably expressing HA-PPP2R5B (upper panels) or HA-APOBEC3G (lower panels) were transduced with the indicated shRNA. Cells were then transfected with NL4-3 Vif and analysed by intracellular flow cytometry for HA. Green/red shading shows Vif-transfected cells in the indicated shRNA background. Red lines showing HA staining in cells transduced with control shRNA are included in each panel for reference. In all experiments, cells were analysed 36 hr post-transfection, and transfected cells determined by co-transfection with GFP ( A and C ) or mCherry ( B ). MFI values are shown for transfected (red/green) and untransfected (blue) cells. DOI: http://dx.doi.org/10.7554/eLife.18296.015

    Journal: eLife

    Article Title: Temporal proteomic analysis of HIV infection reveals remodelling of the host phosphoproteome by lentiviral Vif variants

    doi: 10.7554/eLife.18296

    Figure Lengend Snippet: ( A ) Proteasomal degradation. 293T cells stably expressing HA-PPP2R5A were transfected with NL4-3 Vif in the presence of DMSO (control) or the proteasome inhibitor bortezomib and analysed by intracellular flow cytometry for HA. ( B ) CUL5-dependent degradation. 293T cells stably expressing GFP-PPP2R5B were co-transfected with NL4-3 Vif plus empty vector, wildtype cullin-5 (CUL5 WT) or a dominant negative cullin-5 mutant (CUL5 DN) and analysed by flow cytometry for GFP. ( C ) CUL5 complex-dependent degradation. 293T cells stably expressing HA-PPP2R5B (upper panels) or HA-APOBEC3G (lower panels) were transduced with the indicated shRNA. Cells were then transfected with NL4-3 Vif and analysed by intracellular flow cytometry for HA. Green/red shading shows Vif-transfected cells in the indicated shRNA background. Red lines showing HA staining in cells transduced with control shRNA are included in each panel for reference. In all experiments, cells were analysed 36 hr post-transfection, and transfected cells determined by co-transfection with GFP ( A and C ) or mCherry ( B ). MFI values are shown for transfected (red/green) and untransfected (blue) cells. DOI: http://dx.doi.org/10.7554/eLife.18296.015

    Article Snippet: The dominant negative (DN) CUL5 expression plasmid pcDNA3-DN-hCUL5-FLAG was a kind gift from Prof. Wade Harper (Addgene plasmid #15823 [ ]).

    Techniques: Stable Transfection, Expressing, Transfection, Flow Cytometry, Plasmid Preparation, Dominant Negative Mutation, Mutagenesis, Transduction, shRNA, Staining, Cotransfection