Structured Review

GenScript c terminal flag
Nef binds to Ser5. (A) Schematic of the BiFC fusion proteins is presented. Venus is divided into an N-terminal region from residues 2 to 173 (VN) and a C-terminal region from residues 154 to 238 (VC). VN and VC were fused to the C terminus of Ser5 or Nef, and each fusion protein also contains an HA, V5, or <t>FLAG</t> epitope. (B) pcDNA3.1-Nef-VN-HA, pcDNA3.1-Nef4D-VN-HA, pcDNA3.1-Ser5-VN-HA, pcDNA3.1-Nef-V5-VC, pcDNA3.1-Nef4D-V5-VC, pcDNA3.1-CD4-V5-VC, and pcDNA3.1-Ser5-FLAG-VC were expressed individually or pairwise in 293T cells as indicated. The mean fluorescence intensities (MFI) of BiFC fluorescent signals were determined by flow cytometry and are presented as relative values to the signal from untransfected cells. Error bars represent the SD from three independent experiments. (C) The vectors in panel B are expressed pairwise in HeLa cells as indicated. Ser5-VN-HA (panels I and II) and Nef-VN-HA (panel IV) fusion proteins were stained with a rabbit anti-HA monoclonal antibody at a dilution of 1:500, followed by an Alexa Fluor 647-conjuated donkey anti-rabbit antibody at a 1:1,000 dilution. CD4-V5-VC (panel III) fusion was stained with a mouse anti-V5 antibody at a 1:250 dilution, followed by an Alexa Fluor 647-conjugated goat anti-mouse antibody at a 1:1,000 dilution. Colocalization of the red and the BiFC green fluorescent signals were visualized by confocal microscopy. (D) Schematic of the HIV-1 NL4-3 Nef protein. The N-terminal flexible anchor domain (AN), core domains, internal flexible loop (FL), and critical residues and motifs for MHC-I (red) and CD4 (green) downregulation are shown. The indicated Nef mutations were created in the pcDNA3.1-Nef-V5-VC vector, and Nef expression was determined by Western blotting with an anti-V5 antibody. (E) <t>pMSCV-Ser5-VN-HA</t> was expressed with pcDNA3.1-Nef-V5-VC vectors expressing the indicated Nef mutants in 293T cells. The BiFC MFI was determined by flow cytometry and are presented as values relative to the signal from untransfected cells. Error bars represent the SD from three independent experiments.
C Terminal Flag, supplied by GenScript, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/c terminal flag/product/GenScript
Average 92 stars, based on 1 article reviews
Price from $9.99 to $1999.99
c terminal flag - by Bioz Stars, 2020-09
92/100 stars

Images

1) Product Images from "HIV-1 Nef Antagonizes SERINC5 Restriction by Downregulation of SERINC5 via the Endosome/Lysosome System"

Article Title: HIV-1 Nef Antagonizes SERINC5 Restriction by Downregulation of SERINC5 via the Endosome/Lysosome System

Journal: Journal of Virology

doi: 10.1128/JVI.00196-18

Nef binds to Ser5. (A) Schematic of the BiFC fusion proteins is presented. Venus is divided into an N-terminal region from residues 2 to 173 (VN) and a C-terminal region from residues 154 to 238 (VC). VN and VC were fused to the C terminus of Ser5 or Nef, and each fusion protein also contains an HA, V5, or FLAG epitope. (B) pcDNA3.1-Nef-VN-HA, pcDNA3.1-Nef4D-VN-HA, pcDNA3.1-Ser5-VN-HA, pcDNA3.1-Nef-V5-VC, pcDNA3.1-Nef4D-V5-VC, pcDNA3.1-CD4-V5-VC, and pcDNA3.1-Ser5-FLAG-VC were expressed individually or pairwise in 293T cells as indicated. The mean fluorescence intensities (MFI) of BiFC fluorescent signals were determined by flow cytometry and are presented as relative values to the signal from untransfected cells. Error bars represent the SD from three independent experiments. (C) The vectors in panel B are expressed pairwise in HeLa cells as indicated. Ser5-VN-HA (panels I and II) and Nef-VN-HA (panel IV) fusion proteins were stained with a rabbit anti-HA monoclonal antibody at a dilution of 1:500, followed by an Alexa Fluor 647-conjuated donkey anti-rabbit antibody at a 1:1,000 dilution. CD4-V5-VC (panel III) fusion was stained with a mouse anti-V5 antibody at a 1:250 dilution, followed by an Alexa Fluor 647-conjugated goat anti-mouse antibody at a 1:1,000 dilution. Colocalization of the red and the BiFC green fluorescent signals were visualized by confocal microscopy. (D) Schematic of the HIV-1 NL4-3 Nef protein. The N-terminal flexible anchor domain (AN), core domains, internal flexible loop (FL), and critical residues and motifs for MHC-I (red) and CD4 (green) downregulation are shown. The indicated Nef mutations were created in the pcDNA3.1-Nef-V5-VC vector, and Nef expression was determined by Western blotting with an anti-V5 antibody. (E) pMSCV-Ser5-VN-HA was expressed with pcDNA3.1-Nef-V5-VC vectors expressing the indicated Nef mutants in 293T cells. The BiFC MFI was determined by flow cytometry and are presented as values relative to the signal from untransfected cells. Error bars represent the SD from three independent experiments.
Figure Legend Snippet: Nef binds to Ser5. (A) Schematic of the BiFC fusion proteins is presented. Venus is divided into an N-terminal region from residues 2 to 173 (VN) and a C-terminal region from residues 154 to 238 (VC). VN and VC were fused to the C terminus of Ser5 or Nef, and each fusion protein also contains an HA, V5, or FLAG epitope. (B) pcDNA3.1-Nef-VN-HA, pcDNA3.1-Nef4D-VN-HA, pcDNA3.1-Ser5-VN-HA, pcDNA3.1-Nef-V5-VC, pcDNA3.1-Nef4D-V5-VC, pcDNA3.1-CD4-V5-VC, and pcDNA3.1-Ser5-FLAG-VC were expressed individually or pairwise in 293T cells as indicated. The mean fluorescence intensities (MFI) of BiFC fluorescent signals were determined by flow cytometry and are presented as relative values to the signal from untransfected cells. Error bars represent the SD from three independent experiments. (C) The vectors in panel B are expressed pairwise in HeLa cells as indicated. Ser5-VN-HA (panels I and II) and Nef-VN-HA (panel IV) fusion proteins were stained with a rabbit anti-HA monoclonal antibody at a dilution of 1:500, followed by an Alexa Fluor 647-conjuated donkey anti-rabbit antibody at a 1:1,000 dilution. CD4-V5-VC (panel III) fusion was stained with a mouse anti-V5 antibody at a 1:250 dilution, followed by an Alexa Fluor 647-conjugated goat anti-mouse antibody at a 1:1,000 dilution. Colocalization of the red and the BiFC green fluorescent signals were visualized by confocal microscopy. (D) Schematic of the HIV-1 NL4-3 Nef protein. The N-terminal flexible anchor domain (AN), core domains, internal flexible loop (FL), and critical residues and motifs for MHC-I (red) and CD4 (green) downregulation are shown. The indicated Nef mutations were created in the pcDNA3.1-Nef-V5-VC vector, and Nef expression was determined by Western blotting with an anti-V5 antibody. (E) pMSCV-Ser5-VN-HA was expressed with pcDNA3.1-Nef-V5-VC vectors expressing the indicated Nef mutants in 293T cells. The BiFC MFI was determined by flow cytometry and are presented as values relative to the signal from untransfected cells. Error bars represent the SD from three independent experiments.

Techniques Used: Bimolecular Fluorescence Complementation Assay, FLAG-tag, Fluorescence, Flow Cytometry, Cytometry, Staining, Confocal Microscopy, Plasmid Preparation, Expressing, Western Blot

Role of ubiquitination in Nef-mediated Ser5 downregulation. (A) Ser5, CD4, and MHC-I were expressed with WT ubiquitin (Ub WT ) or its mutants (Ub K48R and Ub K63R ) in the presence or absence of Nef in 293T cells, and protein expression was determined by Western blotting. (B) Ser5 was expressed with the indicated ubiquitin expression vector in the presence or absence of Nef expression in 293T cells. Proteins were immunoprecipitated (IP) by an anti-FLAG antibody that targets Ser5 and analyzed by an anti-HA antibody that targets ubiquitin via Western blotting. (C) pcDNA3.1-Ub-VN-HA was expressed alone or with pcDNA3.1-Ser5-FLAG-VC in HeLa cells in the presence of pNLΔE or pNLΔEΔN. The single Ub expression was detected with an anti-HA antibody, followed by Alexa Fluor 488-conjugated secondary antibodies. Nuclei were stained with DAPI. Fluorescent signals were visualized by confocal microscopy. The colocalization of Ser5 and ubiquitin in the presence or absence of Nef was statistically analyzed. Error bars indicate the SD from three independent experiments.
Figure Legend Snippet: Role of ubiquitination in Nef-mediated Ser5 downregulation. (A) Ser5, CD4, and MHC-I were expressed with WT ubiquitin (Ub WT ) or its mutants (Ub K48R and Ub K63R ) in the presence or absence of Nef in 293T cells, and protein expression was determined by Western blotting. (B) Ser5 was expressed with the indicated ubiquitin expression vector in the presence or absence of Nef expression in 293T cells. Proteins were immunoprecipitated (IP) by an anti-FLAG antibody that targets Ser5 and analyzed by an anti-HA antibody that targets ubiquitin via Western blotting. (C) pcDNA3.1-Ub-VN-HA was expressed alone or with pcDNA3.1-Ser5-FLAG-VC in HeLa cells in the presence of pNLΔE or pNLΔEΔN. The single Ub expression was detected with an anti-HA antibody, followed by Alexa Fluor 488-conjugated secondary antibodies. Nuclei were stained with DAPI. Fluorescent signals were visualized by confocal microscopy. The colocalization of Ser5 and ubiquitin in the presence or absence of Nef was statistically analyzed. Error bars indicate the SD from three independent experiments.

Techniques Used: Expressing, Western Blot, Plasmid Preparation, Immunoprecipitation, Staining, Confocal Microscopy

Nef decreases Ser5 expression at steady-state levels. (A) Wild-type (WT) or Nef-deficient (ΔN) HIV-1 pseudoviruses were produced from 293T cells in the presence of pBJ6-Ser5 or a control (Ctrl) vector, and viral infectivity was determined in TZM-bI cells. Error bars indicate standard deviations (SD) from three independent experiments. (B) pBJ5-iHA-Ser5 and pMSCV-CD4-FLAG were expressed in 293T cells in the presence of a WT or indicated Nef mutant expression vector, and the Ser5 and CD4 expression on cell surface were determined by flow cytometry. Error bars indicate the standard errors of the mean (SEM) from three independent experiments. (C) 293T cells were transfected with pNLΔE or pNLΔEΔN in the presence of increasing amounts of pBJ5-iHA-Ser5. Virions were purified from culture supernatants via ultracentrifugation. Protein expression in cells and virions was analyzed by Western blotting. (D) Ser5 was expressed in 293T cells in the presence or absence of Nef. Cells were treated with DBeQ (15 μM), MG132 (10 μM), NH 4 Cl (20 μM), wortmannin (100 nM), bafilomycin A1 (100 nM), or dimethyl sulfoxide for 24 h, and protein expression was analyzed by Western blotting. (E) MHC-I and CD4 were expressed in 293T cells in the presence or absence of Nef and treated with bafilomycin A1 (100 nM) for 24 h. Protein expression was analyzed by Western blotting.
Figure Legend Snippet: Nef decreases Ser5 expression at steady-state levels. (A) Wild-type (WT) or Nef-deficient (ΔN) HIV-1 pseudoviruses were produced from 293T cells in the presence of pBJ6-Ser5 or a control (Ctrl) vector, and viral infectivity was determined in TZM-bI cells. Error bars indicate standard deviations (SD) from three independent experiments. (B) pBJ5-iHA-Ser5 and pMSCV-CD4-FLAG were expressed in 293T cells in the presence of a WT or indicated Nef mutant expression vector, and the Ser5 and CD4 expression on cell surface were determined by flow cytometry. Error bars indicate the standard errors of the mean (SEM) from three independent experiments. (C) 293T cells were transfected with pNLΔE or pNLΔEΔN in the presence of increasing amounts of pBJ5-iHA-Ser5. Virions were purified from culture supernatants via ultracentrifugation. Protein expression in cells and virions was analyzed by Western blotting. (D) Ser5 was expressed in 293T cells in the presence or absence of Nef. Cells were treated with DBeQ (15 μM), MG132 (10 μM), NH 4 Cl (20 μM), wortmannin (100 nM), bafilomycin A1 (100 nM), or dimethyl sulfoxide for 24 h, and protein expression was analyzed by Western blotting. (E) MHC-I and CD4 were expressed in 293T cells in the presence or absence of Nef and treated with bafilomycin A1 (100 nM) for 24 h. Protein expression was analyzed by Western blotting.

Techniques Used: Expressing, Produced, Plasmid Preparation, Infection, Mutagenesis, Flow Cytometry, Cytometry, Transfection, Purification, Western Blot

Related Articles

Clone Assay:

Article Title: HIV-1 Nef Antagonizes SERINC5 Restriction by Downregulation of SERINC5 via the Endosome/Lysosome System
Article Snippet: .. The Ub K48R, K63R, and K48/63R mutants were created in the pCMV-HA-Ub vector by site-directed mutagenesis. pMSCV-CD4-FLAG expression vector was created by cloning the CD4 cDNA with a C-terminal FLAG into the pMSCVpuro vector after XhoI/EcoRI digestion. pCMV-CD4-FLAG was constructed by cloning CD4 into the pCMV6-Entry vector after BamHI/EcoRV digestion. pcDNA3.1-MHC-I-FLAG expressing the HLA-A gene ( ) from the pcDNA3.1+/C-(K)DYK vector was ordered from GenScript (product no. OHu21196D). pcDNA3.1-SF2Nef-V5 expression vector was created by directly cloning the Nef gene from HIV-1 SF2 strain into the pcDNA3.1D/V5-His-TOPO vector via the TOPO-cloning strategy (Invitrogen). .. SF2Nef G2A, ΔCAW, AxxA, RR/LL, LL/AA, ED/AA, and ΔFL mutants were created by site-directed mutagenesis in the pcDNA3.1-SF2Nef-V5 vector.

Article Title: Reaction mechanisms of Pol IV, RDR2 and DCL3 drive RNA channeling in the siRNA-directed DNA methylation pathway
Article Snippet: .. Cloning, expression, and purification of recombinant DCL3 A DCL3 cDNA, codon optimized for protein expression in insect cells and including a N-terminal 10X His-tag and C-terminal FLAG and Strep tags, was synthesized by GenScript® and cloned into pUC57. .. The cDNA was subcloned into a pFastBacTM HT B vector (Thermo Fisher Scientific) and used to make recombinant bacmid DNA in E coli DH10Bac cells, according to the supplier’s protocol (Bac-to-Bac® Baculovirus expression system; Thermo Fisher Scientific).

Synthesized:

Article Title: Reaction mechanisms of Pol IV, RDR2 and DCL3 drive RNA channeling in the siRNA-directed DNA methylation pathway
Article Snippet: .. Cloning, expression, and purification of recombinant DCL3 A DCL3 cDNA, codon optimized for protein expression in insect cells and including a N-terminal 10X His-tag and C-terminal FLAG and Strep tags, was synthesized by GenScript® and cloned into pUC57. .. The cDNA was subcloned into a pFastBacTM HT B vector (Thermo Fisher Scientific) and used to make recombinant bacmid DNA in E coli DH10Bac cells, according to the supplier’s protocol (Bac-to-Bac® Baculovirus expression system; Thermo Fisher Scientific).

Mutagenesis:

Article Title: HIV-1 Nef Antagonizes SERINC5 Restriction by Downregulation of SERINC5 via the Endosome/Lysosome System
Article Snippet: .. The Ub K48R, K63R, and K48/63R mutants were created in the pCMV-HA-Ub vector by site-directed mutagenesis. pMSCV-CD4-FLAG expression vector was created by cloning the CD4 cDNA with a C-terminal FLAG into the pMSCVpuro vector after XhoI/EcoRI digestion. pCMV-CD4-FLAG was constructed by cloning CD4 into the pCMV6-Entry vector after BamHI/EcoRV digestion. pcDNA3.1-MHC-I-FLAG expressing the HLA-A gene ( ) from the pcDNA3.1+/C-(K)DYK vector was ordered from GenScript (product no. OHu21196D). pcDNA3.1-SF2Nef-V5 expression vector was created by directly cloning the Nef gene from HIV-1 SF2 strain into the pcDNA3.1D/V5-His-TOPO vector via the TOPO-cloning strategy (Invitrogen). .. SF2Nef G2A, ΔCAW, AxxA, RR/LL, LL/AA, ED/AA, and ΔFL mutants were created by site-directed mutagenesis in the pcDNA3.1-SF2Nef-V5 vector.

Construct:

Article Title: HIV-1 Nef Antagonizes SERINC5 Restriction by Downregulation of SERINC5 via the Endosome/Lysosome System
Article Snippet: .. The Ub K48R, K63R, and K48/63R mutants were created in the pCMV-HA-Ub vector by site-directed mutagenesis. pMSCV-CD4-FLAG expression vector was created by cloning the CD4 cDNA with a C-terminal FLAG into the pMSCVpuro vector after XhoI/EcoRI digestion. pCMV-CD4-FLAG was constructed by cloning CD4 into the pCMV6-Entry vector after BamHI/EcoRV digestion. pcDNA3.1-MHC-I-FLAG expressing the HLA-A gene ( ) from the pcDNA3.1+/C-(K)DYK vector was ordered from GenScript (product no. OHu21196D). pcDNA3.1-SF2Nef-V5 expression vector was created by directly cloning the Nef gene from HIV-1 SF2 strain into the pcDNA3.1D/V5-His-TOPO vector via the TOPO-cloning strategy (Invitrogen). .. SF2Nef G2A, ΔCAW, AxxA, RR/LL, LL/AA, ED/AA, and ΔFL mutants were created by site-directed mutagenesis in the pcDNA3.1-SF2Nef-V5 vector.

Purification:

Article Title: Reaction mechanisms of Pol IV, RDR2 and DCL3 drive RNA channeling in the siRNA-directed DNA methylation pathway
Article Snippet: .. Cloning, expression, and purification of recombinant DCL3 A DCL3 cDNA, codon optimized for protein expression in insect cells and including a N-terminal 10X His-tag and C-terminal FLAG and Strep tags, was synthesized by GenScript® and cloned into pUC57. .. The cDNA was subcloned into a pFastBacTM HT B vector (Thermo Fisher Scientific) and used to make recombinant bacmid DNA in E coli DH10Bac cells, according to the supplier’s protocol (Bac-to-Bac® Baculovirus expression system; Thermo Fisher Scientific).

Expressing:

Article Title: HIV-1 Nef Antagonizes SERINC5 Restriction by Downregulation of SERINC5 via the Endosome/Lysosome System
Article Snippet: .. The Ub K48R, K63R, and K48/63R mutants were created in the pCMV-HA-Ub vector by site-directed mutagenesis. pMSCV-CD4-FLAG expression vector was created by cloning the CD4 cDNA with a C-terminal FLAG into the pMSCVpuro vector after XhoI/EcoRI digestion. pCMV-CD4-FLAG was constructed by cloning CD4 into the pCMV6-Entry vector after BamHI/EcoRV digestion. pcDNA3.1-MHC-I-FLAG expressing the HLA-A gene ( ) from the pcDNA3.1+/C-(K)DYK vector was ordered from GenScript (product no. OHu21196D). pcDNA3.1-SF2Nef-V5 expression vector was created by directly cloning the Nef gene from HIV-1 SF2 strain into the pcDNA3.1D/V5-His-TOPO vector via the TOPO-cloning strategy (Invitrogen). .. SF2Nef G2A, ΔCAW, AxxA, RR/LL, LL/AA, ED/AA, and ΔFL mutants were created by site-directed mutagenesis in the pcDNA3.1-SF2Nef-V5 vector.

Article Title: Reaction mechanisms of Pol IV, RDR2 and DCL3 drive RNA channeling in the siRNA-directed DNA methylation pathway
Article Snippet: .. Cloning, expression, and purification of recombinant DCL3 A DCL3 cDNA, codon optimized for protein expression in insect cells and including a N-terminal 10X His-tag and C-terminal FLAG and Strep tags, was synthesized by GenScript® and cloned into pUC57. .. The cDNA was subcloned into a pFastBacTM HT B vector (Thermo Fisher Scientific) and used to make recombinant bacmid DNA in E coli DH10Bac cells, according to the supplier’s protocol (Bac-to-Bac® Baculovirus expression system; Thermo Fisher Scientific).

Sequencing:

Article Title: Molecular characterisation of ILRUN, a novel inhibitor of proinflammatory and antimicrobial cytokines
Article Snippet: .. The ΔNBR1 coding sequence was designed upon the naturally occurring isoform ILRUNb (NCBI NP_073595.2) and was synthesised by GenScript with a C-terminal FLAG coding sequence. .. All amplicons were ligated into the mammalian expression vector pCAGGS using the SacI and XhoI restriction sites and transformed into chemically competent Escherichia coli (MAX Efficiency DH5α competent cells, Thermo Fisher Scientific, Massachusetts, United States).

Recombinant:

Article Title: Reaction mechanisms of Pol IV, RDR2 and DCL3 drive RNA channeling in the siRNA-directed DNA methylation pathway
Article Snippet: .. Cloning, expression, and purification of recombinant DCL3 A DCL3 cDNA, codon optimized for protein expression in insect cells and including a N-terminal 10X His-tag and C-terminal FLAG and Strep tags, was synthesized by GenScript® and cloned into pUC57. .. The cDNA was subcloned into a pFastBacTM HT B vector (Thermo Fisher Scientific) and used to make recombinant bacmid DNA in E coli DH10Bac cells, according to the supplier’s protocol (Bac-to-Bac® Baculovirus expression system; Thermo Fisher Scientific).

Plasmid Preparation:

Article Title: HIV-1 Nef Antagonizes SERINC5 Restriction by Downregulation of SERINC5 via the Endosome/Lysosome System
Article Snippet: .. The Ub K48R, K63R, and K48/63R mutants were created in the pCMV-HA-Ub vector by site-directed mutagenesis. pMSCV-CD4-FLAG expression vector was created by cloning the CD4 cDNA with a C-terminal FLAG into the pMSCVpuro vector after XhoI/EcoRI digestion. pCMV-CD4-FLAG was constructed by cloning CD4 into the pCMV6-Entry vector after BamHI/EcoRV digestion. pcDNA3.1-MHC-I-FLAG expressing the HLA-A gene ( ) from the pcDNA3.1+/C-(K)DYK vector was ordered from GenScript (product no. OHu21196D). pcDNA3.1-SF2Nef-V5 expression vector was created by directly cloning the Nef gene from HIV-1 SF2 strain into the pcDNA3.1D/V5-His-TOPO vector via the TOPO-cloning strategy (Invitrogen). .. SF2Nef G2A, ΔCAW, AxxA, RR/LL, LL/AA, ED/AA, and ΔFL mutants were created by site-directed mutagenesis in the pcDNA3.1-SF2Nef-V5 vector.

Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 91
    GenScript c terminal flag tag
    Design and validation of expression of CoV nsp2 and <t>nsp4</t> constructs for affinity purification. (A) Schematic of SARS-CoV-2 genome organization. (B) Amino acid sequence identity and similarity (in parentheses) for comparisons of nsp2 and nsp4 homologs. Sequence alignments are shown in Figure S1A-B. (C) Nsp2 and nsp4 <t>FLAG-tagged</t> construct designs. Nsp2 constructs contain an N-terminal FLAG-tag. Nsp4 constructs contain a 19 amino acid leader sequence from nsp3 at the N-terminus, including the PL2 pro cleavage site, along with a C-terminal FLAG-tag. (D-E) Western blot of nsp2 and nsp4 homologs expressed in HEK293T cells. Cell were transiently transfected with FLAG-nsp2 (D) or nsp4-FLAG (E). Proteins were detected using an anti-FLAG antibody.
    C Terminal Flag Tag, supplied by GenScript, used in various techniques. Bioz Stars score: 91/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/c terminal flag tag/product/GenScript
    Average 91 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    c terminal flag tag - by Bioz Stars, 2020-09
    91/100 stars
      Buy from Supplier

    84
    GenScript human navβ3
    Effects of SCNA10 variants on Nav1.8 current density and total protein expressing. (A) Representative Nav1.8 current traces of WT, L388M and R756W channel co-expressed <t>Navβ3</t> subunit in HEK293 cells. (B) Representative immunoblot of total proteins from HEK293 cells transfected with WT or mutant Nav1.8. Input lysates were probed with a pan-Nav antibody. GAPDH antibody was used as a protein loading control. (C) The peak currents at 0mV was normalized to the cell surface size by dividing by the cell capacitance and plotted. Compared to WT, the L388M mutant current density was significantly reduced, −11.08 ± 3.66 pA/pF; n = 7 vs −2.43 ± 1.74 pA/pF; n=7. The R756W (−18.94 ± 5.35 pA/pF; n = 5), L867F (−14.55 ± 1.62 pA/pF; n = 5), P1102S (−19.03 ± 4.36 pA/pF; n=5) and V1518I (−12.12 ± 3.28 pA/pF; n=5) mutants all showed non-significant increased peak currents at 0 mV when compared to WT. *P
    Human Navβ3, supplied by GenScript, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/human navβ3/product/GenScript
    Average 84 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    human navβ3 - by Bioz Stars, 2020-09
    84/100 stars
      Buy from Supplier

    Image Search Results


    Design and validation of expression of CoV nsp2 and nsp4 constructs for affinity purification. (A) Schematic of SARS-CoV-2 genome organization. (B) Amino acid sequence identity and similarity (in parentheses) for comparisons of nsp2 and nsp4 homologs. Sequence alignments are shown in Figure S1A-B. (C) Nsp2 and nsp4 FLAG-tagged construct designs. Nsp2 constructs contain an N-terminal FLAG-tag. Nsp4 constructs contain a 19 amino acid leader sequence from nsp3 at the N-terminus, including the PL2 pro cleavage site, along with a C-terminal FLAG-tag. (D-E) Western blot of nsp2 and nsp4 homologs expressed in HEK293T cells. Cell were transiently transfected with FLAG-nsp2 (D) or nsp4-FLAG (E). Proteins were detected using an anti-FLAG antibody.

    Journal: bioRxiv

    Article Title: Comparative multiplexed interactomics of SARS-CoV-2 and homologous coronavirus non-structural proteins identifies unique and shared host-cell dependencies

    doi: 10.1101/2020.07.13.201517

    Figure Lengend Snippet: Design and validation of expression of CoV nsp2 and nsp4 constructs for affinity purification. (A) Schematic of SARS-CoV-2 genome organization. (B) Amino acid sequence identity and similarity (in parentheses) for comparisons of nsp2 and nsp4 homologs. Sequence alignments are shown in Figure S1A-B. (C) Nsp2 and nsp4 FLAG-tagged construct designs. Nsp2 constructs contain an N-terminal FLAG-tag. Nsp4 constructs contain a 19 amino acid leader sequence from nsp3 at the N-terminus, including the PL2 pro cleavage site, along with a C-terminal FLAG-tag. (D-E) Western blot of nsp2 and nsp4 homologs expressed in HEK293T cells. Cell were transiently transfected with FLAG-nsp2 (D) or nsp4-FLAG (E). Proteins were detected using an anti-FLAG antibody.

    Article Snippet: Human codon optimized sequences were designed, genes synthesized, and cloned into pcDNA3.1-(+)-C-DYK (nsp4) to append a C-terminal FLAG tag, or into pcDNA3.1-(+)-N-DYK (nsp2) to append an N-terminal FLAG tag (GenScript).

    Techniques: Expressing, Construct, Affinity Purification, Sequencing, FLAG-tag, Western Blot, Transfection

    Affinity purification-mass spectrometry (AP-MS) identifies nsp2 interactors. (A) General AP-MS workflow to quantitatively determine interactors of viral nsp homolog. HEK293T cells are transfected with FLAG-tagged expression constructs of nsps as bait or GFP (mock) and lysed. Bait proteins are immunoprecipitated (IP) along with interacting proteins, reduced, alkylated, and tryptic digested. Peptides are then tandem-mass tag (TMT) labeled, pooled, and analyzed by LC-MS/MS for identification and quantification. (B) Data processing workflow. Peptide spectra are identified and matched to corresponding proteins (SEQUEST HT), then quantified based on TMT reporter ion intensity (Proteome Discoverer 2.4). High confidence interactors are filtered by comparing bait vs control. Interaction ratios between bait homologs are determined (log2 fold change) and adjusted p-value calculated using ANOVA. (C-D) Volcano plot of SARS-CoV-2 nsp2 (C) and SARS-CoV-1 nsp2 (D) datasets to identify medium- and high-confidence interactors. Plotted are log2 TMT intensity fold changes for proteins between nsp2 bait channels and GFP mock transfections versus -log10 adjusted p-values. Curves for the variable cutoffs used to define high-confidence (red) or medium confidence (blue) interactors are shown. 1σ = 0.5 for (C), 1σ = 0.43 for (D). (E) Venn diagram comparing high-confidence interactors between nsp2 homologs. Sixteen unique proteins were identified each, while four proteins overlapped both data sets (listed in adjacent table).

    Journal: bioRxiv

    Article Title: Comparative multiplexed interactomics of SARS-CoV-2 and homologous coronavirus non-structural proteins identifies unique and shared host-cell dependencies

    doi: 10.1101/2020.07.13.201517

    Figure Lengend Snippet: Affinity purification-mass spectrometry (AP-MS) identifies nsp2 interactors. (A) General AP-MS workflow to quantitatively determine interactors of viral nsp homolog. HEK293T cells are transfected with FLAG-tagged expression constructs of nsps as bait or GFP (mock) and lysed. Bait proteins are immunoprecipitated (IP) along with interacting proteins, reduced, alkylated, and tryptic digested. Peptides are then tandem-mass tag (TMT) labeled, pooled, and analyzed by LC-MS/MS for identification and quantification. (B) Data processing workflow. Peptide spectra are identified and matched to corresponding proteins (SEQUEST HT), then quantified based on TMT reporter ion intensity (Proteome Discoverer 2.4). High confidence interactors are filtered by comparing bait vs control. Interaction ratios between bait homologs are determined (log2 fold change) and adjusted p-value calculated using ANOVA. (C-D) Volcano plot of SARS-CoV-2 nsp2 (C) and SARS-CoV-1 nsp2 (D) datasets to identify medium- and high-confidence interactors. Plotted are log2 TMT intensity fold changes for proteins between nsp2 bait channels and GFP mock transfections versus -log10 adjusted p-values. Curves for the variable cutoffs used to define high-confidence (red) or medium confidence (blue) interactors are shown. 1σ = 0.5 for (C), 1σ = 0.43 for (D). (E) Venn diagram comparing high-confidence interactors between nsp2 homologs. Sixteen unique proteins were identified each, while four proteins overlapped both data sets (listed in adjacent table).

    Article Snippet: Human codon optimized sequences were designed, genes synthesized, and cloned into pcDNA3.1-(+)-C-DYK (nsp4) to append a C-terminal FLAG tag, or into pcDNA3.1-(+)-N-DYK (nsp2) to append an N-terminal FLAG tag (GenScript).

    Techniques: Affinity Purification, Mass Spectrometry, Transfection, Expressing, Construct, Immunoprecipitation, Labeling, Liquid Chromatography with Mass Spectroscopy

    Expression of SARS-CoV-2 VLPs in mammalian expression system. Expression of four structural proteins analyzed using collected cell culture supernatant and cell lysate 48 h after transfection or co-transfection of plasmids encoding S, M, E, or N in both HEK-293T (A,C,E) and Vero E6 cells (B,D,F) . In single plasmid transfection, a total of 2.5 μg of each plasmid was applied to HEK-293T or Vero E6 cells individually. For double and triple plasmids co-transfection, S, M, E, or N plasmid was introduced at equal molar ratio. Construction of SARS-CoV-2 VLPs was performed by co-transfecting cells with S, M, E, and N with molar ratio at 8:6:8:3. The overall expression profile of four structural proteins was visualized using anti-flag antibody (A,B) . Anti-S (C,D) and anti-N antibodies (E,F) were additionally used to confirm expression of S and N, which could not be clearly distinguished by anti-Flag antibody. (G–I) , resuspended pellets from ultracentrifuged cell medium were then loaded on a 20–60% discontinuous sucrose gradient and subjected to fractions by ultracentrifugation. Nineteen fractions were collected (1–19, from lightest to heaviest). The nature of viral proteins associated with each fraction was determined by anti-Flag (G) , anti-S (H) and anti-N (I) antibodies. S, full-length spike protein; S2, membrane fusion subunit of spike protein; Sc, a cleaved membrane fusion subunit of spike protein. Protein marker and negative control (Blank) are shown as indicated.

    Journal: Frontiers in Bioengineering and Biotechnology

    Article Title: Construction of SARS-CoV-2 Virus-Like Particles by Mammalian Expression System

    doi: 10.3389/fbioe.2020.00862

    Figure Lengend Snippet: Expression of SARS-CoV-2 VLPs in mammalian expression system. Expression of four structural proteins analyzed using collected cell culture supernatant and cell lysate 48 h after transfection or co-transfection of plasmids encoding S, M, E, or N in both HEK-293T (A,C,E) and Vero E6 cells (B,D,F) . In single plasmid transfection, a total of 2.5 μg of each plasmid was applied to HEK-293T or Vero E6 cells individually. For double and triple plasmids co-transfection, S, M, E, or N plasmid was introduced at equal molar ratio. Construction of SARS-CoV-2 VLPs was performed by co-transfecting cells with S, M, E, and N with molar ratio at 8:6:8:3. The overall expression profile of four structural proteins was visualized using anti-flag antibody (A,B) . Anti-S (C,D) and anti-N antibodies (E,F) were additionally used to confirm expression of S and N, which could not be clearly distinguished by anti-Flag antibody. (G–I) , resuspended pellets from ultracentrifuged cell medium were then loaded on a 20–60% discontinuous sucrose gradient and subjected to fractions by ultracentrifugation. Nineteen fractions were collected (1–19, from lightest to heaviest). The nature of viral proteins associated with each fraction was determined by anti-Flag (G) , anti-S (H) and anti-N (I) antibodies. S, full-length spike protein; S2, membrane fusion subunit of spike protein; Sc, a cleaved membrane fusion subunit of spike protein. Protein marker and negative control (Blank) are shown as indicated.

    Article Snippet: Plasmid Construction and Molecular Cloning Human codon optimized sequences of genes encoding S, M, E and N structural proteins of SARS-CoV-2 with C-terminal FLAG tag (peptide sequence: DYKDDDDK) were synthesized by Genscript Biotechnology (Nanjing, China): the major structural S glycoprotein (Gen Bank: QHD43416.1), E protein (Gen Bank: QHD43418.1), M protein (Gen Bank: QHD43419.1) and N protein (Gen Bank: QHD43423.2).

    Techniques: Expressing, Cell Culture, Transfection, Cotransfection, Plasmid Preparation, Marker, Negative Control

    Enzymatic synthesis of PA by the AGPAT2 V67M/V167A double mutant. (A) The ER-integral membrane protein AGPAT2 catalyzes the conversion of LPA into PA. (B) PA is synthesized by immunopurified recombinant AGPAT2-FLAG by incorporating oleoyl-CoA into LPA. All assays were performed in quadruple. The results are indicated as averages of the replicates ± SEM. Student’s t -tests were used. ND, not determinable. A statistically significant difference was defined as a p -value of

    Journal: Frontiers in Physiology

    Article Title: A Single Complex Agpat2 Allele in a Patient With Partial Lipodystrophy

    doi: 10.3389/fphys.2018.01363

    Figure Lengend Snippet: Enzymatic synthesis of PA by the AGPAT2 V67M/V167A double mutant. (A) The ER-integral membrane protein AGPAT2 catalyzes the conversion of LPA into PA. (B) PA is synthesized by immunopurified recombinant AGPAT2-FLAG by incorporating oleoyl-CoA into LPA. All assays were performed in quadruple. The results are indicated as averages of the replicates ± SEM. Student’s t -tests were used. ND, not determinable. A statistically significant difference was defined as a p -value of

    Article Snippet: A pCDNA3.1 vector containing the complete coding region of human AGPAT2 and C-terminal FLAG tag (Genscript) was used as a template to generate V67M, V167A, and V67M/V167A using the QuikChange mutagenesis kit (Stratagene) following the manufacturer’s instructions.

    Techniques: Mutagenesis, Synthesized, Recombinant

    Effects of SCNA10 variants on Nav1.8 current density and total protein expressing. (A) Representative Nav1.8 current traces of WT, L388M and R756W channel co-expressed Navβ3 subunit in HEK293 cells. (B) Representative immunoblot of total proteins from HEK293 cells transfected with WT or mutant Nav1.8. Input lysates were probed with a pan-Nav antibody. GAPDH antibody was used as a protein loading control. (C) The peak currents at 0mV was normalized to the cell surface size by dividing by the cell capacitance and plotted. Compared to WT, the L388M mutant current density was significantly reduced, −11.08 ± 3.66 pA/pF; n = 7 vs −2.43 ± 1.74 pA/pF; n=7. The R756W (−18.94 ± 5.35 pA/pF; n = 5), L867F (−14.55 ± 1.62 pA/pF; n = 5), P1102S (−19.03 ± 4.36 pA/pF; n=5) and V1518I (−12.12 ± 3.28 pA/pF; n=5) mutants all showed non-significant increased peak currents at 0 mV when compared to WT. *P

    Journal: Forensic science international

    Article Title: Functional Characterization of SCN10A Variants in Several Cases of Sudden Unexplained Death

    doi: 10.1016/j.forsciint.2019.05.042

    Figure Lengend Snippet: Effects of SCNA10 variants on Nav1.8 current density and total protein expressing. (A) Representative Nav1.8 current traces of WT, L388M and R756W channel co-expressed Navβ3 subunit in HEK293 cells. (B) Representative immunoblot of total proteins from HEK293 cells transfected with WT or mutant Nav1.8. Input lysates were probed with a pan-Nav antibody. GAPDH antibody was used as a protein loading control. (C) The peak currents at 0mV was normalized to the cell surface size by dividing by the cell capacitance and plotted. Compared to WT, the L388M mutant current density was significantly reduced, −11.08 ± 3.66 pA/pF; n = 7 vs −2.43 ± 1.74 pA/pF; n=7. The R756W (−18.94 ± 5.35 pA/pF; n = 5), L867F (−14.55 ± 1.62 pA/pF; n = 5), P1102S (−19.03 ± 4.36 pA/pF; n=5) and V1518I (−12.12 ± 3.28 pA/pF; n=5) mutants all showed non-significant increased peak currents at 0 mV when compared to WT. *P

    Article Snippet: The human Navβ3 (Genbank , with C-terminal flag epitope) cDNA in the pcDNA3.1(+) vector was obtained from GenScript (Product ID: OHu16302D).

    Techniques: Expressing, Transfection, Mutagenesis

    Effects of SCNA10 variants on the rate of inactivation of Nav1.8 currents (A) Representative Nav1.8 current traces of WT and P1102S channels co-expressed Navβ3 subunit in HEK293 cells. The recordings were made at 0 mV. (B) The inactivating portions of currents at −20 to +20 mV were subjected to curve fitting to a single exponential function and time constants are plotted as a function of the voltage. *P

    Journal: Forensic science international

    Article Title: Functional Characterization of SCN10A Variants in Several Cases of Sudden Unexplained Death

    doi: 10.1016/j.forsciint.2019.05.042

    Figure Lengend Snippet: Effects of SCNA10 variants on the rate of inactivation of Nav1.8 currents (A) Representative Nav1.8 current traces of WT and P1102S channels co-expressed Navβ3 subunit in HEK293 cells. The recordings were made at 0 mV. (B) The inactivating portions of currents at −20 to +20 mV were subjected to curve fitting to a single exponential function and time constants are plotted as a function of the voltage. *P

    Article Snippet: The human Navβ3 (Genbank , with C-terminal flag epitope) cDNA in the pcDNA3.1(+) vector was obtained from GenScript (Product ID: OHu16302D).

    Techniques:

    Representative Nav1.8 current traces of WT channel expressed in HEK293 in the absence (A) or presence (B) of Navβ3 subunit, using the voltage protocol as shown inset in (C). The Nav1.8 current densities are plotted as a function of the test voltage (C). Data points represent mean ± SEM, with n = 7 in absence and n = 7 in the presence of Navβ3. *P

    Journal: Forensic science international

    Article Title: Functional Characterization of SCN10A Variants in Several Cases of Sudden Unexplained Death

    doi: 10.1016/j.forsciint.2019.05.042

    Figure Lengend Snippet: Representative Nav1.8 current traces of WT channel expressed in HEK293 in the absence (A) or presence (B) of Navβ3 subunit, using the voltage protocol as shown inset in (C). The Nav1.8 current densities are plotted as a function of the test voltage (C). Data points represent mean ± SEM, with n = 7 in absence and n = 7 in the presence of Navβ3. *P

    Article Snippet: The human Navβ3 (Genbank , with C-terminal flag epitope) cDNA in the pcDNA3.1(+) vector was obtained from GenScript (Product ID: OHu16302D).

    Techniques: