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  • 99
    New England Biolabs gibson assembly kit
    Gibson Assembly Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 2061 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    gibson assembly kit - by Bioz Stars, 2020-08
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    92
    GenScript synthetic double stranded dna fragments
    Control experiments for yeast VivosX. ( A,B ) Immunoblots showing the different effects of the FLAG and <t>V5</t> epitope tags on the SDS-PAGE mobility of the H2A.Z and H2A histones. ( C ) <t>DNA</t> samples purified from chromatin isolated from the indicated strains before and after MNase digestion were analyzed by agarose gel electrophoresis and SYBR Green staining. 1x MNase represents 0.08 U/µL of MNase. Circled lanes: MNase digestion conditions selected for the experiment in Figure 3D . ( D,E ) Logarithmically growing HTZ1(T46C) FL cells were fixed with sodium azide (0.1%) for 15 min on ice before treatment with and without 4-DPS (180 µM) for 20 min. The cells were then fixed with TCA fixation and the proteins extracted and analyzed as described for Figure 1D .
    Synthetic Double Stranded Dna Fragments, supplied by GenScript, used in various techniques. Bioz Stars score: 92/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    GenScript codon optimized version
    Control experiments for yeast VivosX. ( A,B ) Immunoblots showing the different effects of the FLAG and <t>V5</t> epitope tags on the SDS-PAGE mobility of the H2A.Z and H2A histones. ( C ) <t>DNA</t> samples purified from chromatin isolated from the indicated strains before and after MNase digestion were analyzed by agarose gel electrophoresis and SYBR Green staining. 1x MNase represents 0.08 U/µL of MNase. Circled lanes: MNase digestion conditions selected for the experiment in Figure 3D . ( D,E ) Logarithmically growing HTZ1(T46C) FL cells were fixed with sodium azide (0.1%) for 15 min on ice before treatment with and without 4-DPS (180 µM) for 20 min. The cells were then fixed with TCA fixation and the proteins extracted and analyzed as described for Figure 1D .
    Codon Optimized Version, supplied by GenScript, used in various techniques. Bioz Stars score: 91/100, based on 293 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    codon optimized version - by Bioz Stars, 2020-08
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    91
    GenScript adp1 genome
    (A) Growth (represented as OD 600 ) of the strains W1 and <t>ADP1</t> WT in 10 and 20 ​g/L casein amino acids. (B) Comparison of WE content between W1 and ADP1 WT in the exponential phase when grown in 10 and 20 ​g/L casein amino acids. Cells of both strains were harvested during exponential phase; ADP1 WT was harvested after 4 ​h, and WI was harvested after 11 ​h. Comparison of (C) CDW and (D) WE content between W1 and ADP1 when cultivated with 20 ​g/L yeast extract. The results represent the mean of two replicates and the error bars represent the standard deviations. ∗∗ P ​
    Adp1 Genome, supplied by GenScript, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    GenScript wild type adenovirus
    (A) Growth (represented as OD 600 ) of the strains W1 and <t>ADP1</t> WT in 10 and 20 ​g/L casein amino acids. (B) Comparison of WE content between W1 and ADP1 WT in the exponential phase when grown in 10 and 20 ​g/L casein amino acids. Cells of both strains were harvested during exponential phase; ADP1 WT was harvested after 4 ​h, and WI was harvested after 11 ​h. Comparison of (C) CDW and (D) WE content between W1 and ADP1 when cultivated with 20 ​g/L yeast extract. The results represent the mean of two replicates and the error bars represent the standard deviations. ∗∗ P ​
    Wild Type Adenovirus, supplied by GenScript, used in various techniques. Bioz Stars score: 90/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    GenScript sacii
    (A) Growth (represented as OD 600 ) of the strains W1 and <t>ADP1</t> WT in 10 and 20 ​g/L casein amino acids. (B) Comparison of WE content between W1 and ADP1 WT in the exponential phase when grown in 10 and 20 ​g/L casein amino acids. Cells of both strains were harvested during exponential phase; ADP1 WT was harvested after 4 ​h, and WI was harvested after 11 ​h. Comparison of (C) CDW and (D) WE content between W1 and ADP1 when cultivated with 20 ​g/L yeast extract. The results represent the mean of two replicates and the error bars represent the standard deviations. ∗∗ P ​
    Sacii, supplied by GenScript, used in various techniques. Bioz Stars score: 92/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    GenScript ecorv
    (A) Growth (represented as OD 600 ) of the strains W1 and <t>ADP1</t> WT in 10 and 20 ​g/L casein amino acids. (B) Comparison of WE content between W1 and ADP1 WT in the exponential phase when grown in 10 and 20 ​g/L casein amino acids. Cells of both strains were harvested during exponential phase; ADP1 WT was harvested after 4 ​h, and WI was harvested after 11 ​h. Comparison of (C) CDW and (D) WE content between W1 and ADP1 when cultivated with 20 ​g/L yeast extract. The results represent the mean of two replicates and the error bars represent the standard deviations. ∗∗ P ​
    Ecorv, supplied by GenScript, used in various techniques. Bioz Stars score: 92/100, based on 42 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    GenScript i scei 18 bp recognition sequence
    (A) Growth (represented as OD 600 ) of the strains W1 and <t>ADP1</t> WT in 10 and 20 ​g/L casein amino acids. (B) Comparison of WE content between W1 and ADP1 WT in the exponential phase when grown in 10 and 20 ​g/L casein amino acids. Cells of both strains were harvested during exponential phase; ADP1 WT was harvested after 4 ​h, and WI was harvested after 11 ​h. Comparison of (C) CDW and (D) WE content between W1 and ADP1 when cultivated with 20 ​g/L yeast extract. The results represent the mean of two replicates and the error bars represent the standard deviations. ∗∗ P ​
    I Scei 18 Bp Recognition Sequence, supplied by GenScript, used in various techniques. Bioz Stars score: 86/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    GenScript δcsd
    (A) Growth (represented as OD 600 ) of the strains W1 and <t>ADP1</t> WT in 10 and 20 ​g/L casein amino acids. (B) Comparison of WE content between W1 and ADP1 WT in the exponential phase when grown in 10 and 20 ​g/L casein amino acids. Cells of both strains were harvested during exponential phase; ADP1 WT was harvested after 4 ​h, and WI was harvested after 11 ​h. Comparison of (C) CDW and (D) WE content between W1 and ADP1 when cultivated with 20 ​g/L yeast extract. The results represent the mean of two replicates and the error bars represent the standard deviations. ∗∗ P ​
    δcsd, supplied by GenScript, used in various techniques. Bioz Stars score: 93/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    GenScript pcr2 1 tetr
    (A) Growth (represented as OD 600 ) of the strains W1 and <t>ADP1</t> WT in 10 and 20 ​g/L casein amino acids. (B) Comparison of WE content between W1 and ADP1 WT in the exponential phase when grown in 10 and 20 ​g/L casein amino acids. Cells of both strains were harvested during exponential phase; ADP1 WT was harvested after 4 ​h, and WI was harvested after 11 ​h. Comparison of (C) CDW and (D) WE content between W1 and ADP1 when cultivated with 20 ​g/L yeast extract. The results represent the mean of two replicates and the error bars represent the standard deviations. ∗∗ P ​
    Pcr2 1 Tetr, supplied by GenScript, used in various techniques. Bioz Stars score: 89/100, based on 100 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    GenScript luxr gene
    (A) Growth (represented as OD 600 ) of the strains W1 and <t>ADP1</t> WT in 10 and 20 ​g/L casein amino acids. (B) Comparison of WE content between W1 and ADP1 WT in the exponential phase when grown in 10 and 20 ​g/L casein amino acids. Cells of both strains were harvested during exponential phase; ADP1 WT was harvested after 4 ​h, and WI was harvested after 11 ​h. Comparison of (C) CDW and (D) WE content between W1 and ADP1 when cultivated with 20 ​g/L yeast extract. The results represent the mean of two replicates and the error bars represent the standard deviations. ∗∗ P ​
    Luxr Gene, supplied by GenScript, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    GenScript pet52b
    (A) Growth (represented as OD 600 ) of the strains W1 and <t>ADP1</t> WT in 10 and 20 ​g/L casein amino acids. (B) Comparison of WE content between W1 and ADP1 WT in the exponential phase when grown in 10 and 20 ​g/L casein amino acids. Cells of both strains were harvested during exponential phase; ADP1 WT was harvested after 4 ​h, and WI was harvested after 11 ​h. Comparison of (C) CDW and (D) WE content between W1 and ADP1 when cultivated with 20 ​g/L yeast extract. The results represent the mean of two replicates and the error bars represent the standard deviations. ∗∗ P ​
    Pet52b, supplied by GenScript, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    ATCC adenovirus generation ad wt
    (A) Growth (represented as OD 600 ) of the strains W1 and <t>ADP1</t> WT in 10 and 20 ​g/L casein amino acids. (B) Comparison of WE content between W1 and ADP1 WT in the exponential phase when grown in 10 and 20 ​g/L casein amino acids. Cells of both strains were harvested during exponential phase; ADP1 WT was harvested after 4 ​h, and WI was harvested after 11 ​h. Comparison of (C) CDW and (D) WE content between W1 and ADP1 when cultivated with 20 ​g/L yeast extract. The results represent the mean of two replicates and the error bars represent the standard deviations. ∗∗ P ​
    Adenovirus Generation Ad Wt, supplied by ATCC, used in various techniques. Bioz Stars score: 90/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    GenScript ncbi databases
    (A) Growth (represented as OD 600 ) of the strains W1 and <t>ADP1</t> WT in 10 and 20 ​g/L casein amino acids. (B) Comparison of WE content between W1 and ADP1 WT in the exponential phase when grown in 10 and 20 ​g/L casein amino acids. Cells of both strains were harvested during exponential phase; ADP1 WT was harvested after 4 ​h, and WI was harvested after 11 ​h. Comparison of (C) CDW and (D) WE content between W1 and ADP1 when cultivated with 20 ​g/L yeast extract. The results represent the mean of two replicates and the error bars represent the standard deviations. ∗∗ P ​
    Ncbi Databases, supplied by GenScript, used in various techniques. Bioz Stars score: 90/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    GenScript puc57
    (A) Growth (represented as OD 600 ) of the strains W1 and <t>ADP1</t> WT in 10 and 20 ​g/L casein amino acids. (B) Comparison of WE content between W1 and ADP1 WT in the exponential phase when grown in 10 and 20 ​g/L casein amino acids. Cells of both strains were harvested during exponential phase; ADP1 WT was harvested after 4 ​h, and WI was harvested after 11 ​h. Comparison of (C) CDW and (D) WE content between W1 and ADP1 when cultivated with 20 ​g/L yeast extract. The results represent the mean of two replicates and the error bars represent the standard deviations. ∗∗ P ​
    Puc57, supplied by GenScript, used in various techniques. Bioz Stars score: 93/100, based on 1626 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    GenScript sb100x
    (A) Growth (represented as OD 600 ) of the strains W1 and <t>ADP1</t> WT in 10 and 20 ​g/L casein amino acids. (B) Comparison of WE content between W1 and ADP1 WT in the exponential phase when grown in 10 and 20 ​g/L casein amino acids. Cells of both strains were harvested during exponential phase; ADP1 WT was harvested after 4 ​h, and WI was harvested after 11 ​h. Comparison of (C) CDW and (D) WE content between W1 and ADP1 when cultivated with 20 ​g/L yeast extract. The results represent the mean of two replicates and the error bars represent the standard deviations. ∗∗ P ​
    Sb100x, supplied by GenScript, used in various techniques. Bioz Stars score: 88/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    GenScript 932 bp dna fragment
    (A) Growth (represented as OD 600 ) of the strains W1 and <t>ADP1</t> WT in 10 and 20 ​g/L casein amino acids. (B) Comparison of WE content between W1 and ADP1 WT in the exponential phase when grown in 10 and 20 ​g/L casein amino acids. Cells of both strains were harvested during exponential phase; ADP1 WT was harvested after 4 ​h, and WI was harvested after 11 ​h. Comparison of (C) CDW and (D) WE content between W1 and ADP1 when cultivated with 20 ​g/L yeast extract. The results represent the mean of two replicates and the error bars represent the standard deviations. ∗∗ P ​
    932 Bp Dna Fragment, supplied by GenScript, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Valiant adenovatorcmv5 shuttle vector
    (A) Growth (represented as OD 600 ) of the strains W1 and <t>ADP1</t> WT in 10 and 20 ​g/L casein amino acids. (B) Comparison of WE content between W1 and ADP1 WT in the exponential phase when grown in 10 and 20 ​g/L casein amino acids. Cells of both strains were harvested during exponential phase; ADP1 WT was harvested after 4 ​h, and WI was harvested after 11 ​h. Comparison of (C) CDW and (D) WE content between W1 and ADP1 when cultivated with 20 ​g/L yeast extract. The results represent the mean of two replicates and the error bars represent the standard deviations. ∗∗ P ​
    Adenovatorcmv5 Shuttle Vector, supplied by Valiant, used in various techniques. Bioz Stars score: 85/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    GenScript n terminal amphipathic alpha helix
    (A) Growth (represented as OD 600 ) of the strains W1 and <t>ADP1</t> WT in 10 and 20 ​g/L casein amino acids. (B) Comparison of WE content between W1 and ADP1 WT in the exponential phase when grown in 10 and 20 ​g/L casein amino acids. Cells of both strains were harvested during exponential phase; ADP1 WT was harvested after 4 ​h, and WI was harvested after 11 ​h. Comparison of (C) CDW and (D) WE content between W1 and ADP1 when cultivated with 20 ​g/L yeast extract. The results represent the mean of two replicates and the error bars represent the standard deviations. ∗∗ P ​
    N Terminal Amphipathic Alpha Helix, 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
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    89
    Stratagene e coli bj5183 cells
    (A) Growth (represented as OD 600 ) of the strains W1 and <t>ADP1</t> WT in 10 and 20 ​g/L casein amino acids. (B) Comparison of WE content between W1 and ADP1 WT in the exponential phase when grown in 10 and 20 ​g/L casein amino acids. Cells of both strains were harvested during exponential phase; ADP1 WT was harvested after 4 ​h, and WI was harvested after 11 ​h. Comparison of (C) CDW and (D) WE content between W1 and ADP1 when cultivated with 20 ​g/L yeast extract. The results represent the mean of two replicates and the error bars represent the standard deviations. ∗∗ P ​
    E Coli Bj5183 Cells, supplied by Stratagene, used in various techniques. Bioz Stars score: 89/100, based on 161 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Stratagene homologous recombination
    (A) Growth (represented as OD 600 ) of the strains W1 and <t>ADP1</t> WT in 10 and 20 ​g/L casein amino acids. (B) Comparison of WE content between W1 and ADP1 WT in the exponential phase when grown in 10 and 20 ​g/L casein amino acids. Cells of both strains were harvested during exponential phase; ADP1 WT was harvested after 4 ​h, and WI was harvested after 11 ​h. Comparison of (C) CDW and (D) WE content between W1 and ADP1 when cultivated with 20 ​g/L yeast extract. The results represent the mean of two replicates and the error bars represent the standard deviations. ∗∗ P ​
    Homologous Recombination, supplied by Stratagene, used in various techniques. Bioz Stars score: 93/100, based on 80 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    GenScript wildtype human tlk1b gene
    (A) Growth (represented as OD 600 ) of the strains W1 and <t>ADP1</t> WT in 10 and 20 ​g/L casein amino acids. (B) Comparison of WE content between W1 and ADP1 WT in the exponential phase when grown in 10 and 20 ​g/L casein amino acids. Cells of both strains were harvested during exponential phase; ADP1 WT was harvested after 4 ​h, and WI was harvested after 11 ​h. Comparison of (C) CDW and (D) WE content between W1 and ADP1 when cultivated with 20 ​g/L yeast extract. The results represent the mean of two replicates and the error bars represent the standard deviations. ∗∗ P ​
    Wildtype Human Tlk1b Gene, supplied by GenScript, used in various techniques. Bioz Stars score: 92/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Thermo Fisher topo cloning strategy
    (A) Growth (represented as OD 600 ) of the strains W1 and <t>ADP1</t> WT in 10 and 20 ​g/L casein amino acids. (B) Comparison of WE content between W1 and ADP1 WT in the exponential phase when grown in 10 and 20 ​g/L casein amino acids. Cells of both strains were harvested during exponential phase; ADP1 WT was harvested after 4 ​h, and WI was harvested after 11 ​h. Comparison of (C) CDW and (D) WE content between W1 and ADP1 when cultivated with 20 ​g/L yeast extract. The results represent the mean of two replicates and the error bars represent the standard deviations. ∗∗ P ​
    Topo Cloning Strategy, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 52 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    GenScript pcdna3 1 c k dyk vector
    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.
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    Control experiments for yeast VivosX. ( A,B ) Immunoblots showing the different effects of the FLAG and V5 epitope tags on the SDS-PAGE mobility of the H2A.Z and H2A histones. ( C ) DNA samples purified from chromatin isolated from the indicated strains before and after MNase digestion were analyzed by agarose gel electrophoresis and SYBR Green staining. 1x MNase represents 0.08 U/µL of MNase. Circled lanes: MNase digestion conditions selected for the experiment in Figure 3D . ( D,E ) Logarithmically growing HTZ1(T46C) FL cells were fixed with sodium azide (0.1%) for 15 min on ice before treatment with and without 4-DPS (180 µM) for 20 min. The cells were then fixed with TCA fixation and the proteins extracted and analyzed as described for Figure 1D .

    Journal: eLife

    Article Title: VivosX, a disulfide crosslinking method to capture site-specific, protein-protein interactions in yeast and human cells

    doi: 10.7554/eLife.36654

    Figure Lengend Snippet: Control experiments for yeast VivosX. ( A,B ) Immunoblots showing the different effects of the FLAG and V5 epitope tags on the SDS-PAGE mobility of the H2A.Z and H2A histones. ( C ) DNA samples purified from chromatin isolated from the indicated strains before and after MNase digestion were analyzed by agarose gel electrophoresis and SYBR Green staining. 1x MNase represents 0.08 U/µL of MNase. Circled lanes: MNase digestion conditions selected for the experiment in Figure 3D . ( D,E ) Logarithmically growing HTZ1(T46C) FL cells were fixed with sodium azide (0.1%) for 15 min on ice before treatment with and without 4-DPS (180 µM) for 20 min. The cells were then fixed with TCA fixation and the proteins extracted and analyzed as described for Figure 1D .

    Article Snippet: For the other V5 HTA1(Cys)-HTB1 plasmids, pEL305 was linearized with BstAPI and MfeI, and synthetic double-stranded DNA fragments (GenScript ) containing individual cysteine substitution at L1 with at least 60 bp of overlapping regions on both sides were recombined using the Gibson assembly kit (New England Biolabs ), creating pEL558-pEL561.

    Techniques: Western Blot, SDS Page, Purification, Isolation, Agarose Gel Electrophoresis, SYBR Green Assay, Staining

    (A) Growth (represented as OD 600 ) of the strains W1 and ADP1 WT in 10 and 20 ​g/L casein amino acids. (B) Comparison of WE content between W1 and ADP1 WT in the exponential phase when grown in 10 and 20 ​g/L casein amino acids. Cells of both strains were harvested during exponential phase; ADP1 WT was harvested after 4 ​h, and WI was harvested after 11 ​h. Comparison of (C) CDW and (D) WE content between W1 and ADP1 when cultivated with 20 ​g/L yeast extract. The results represent the mean of two replicates and the error bars represent the standard deviations. ∗∗ P ​

    Journal: Metabolic Engineering Communications

    Article Title: Wax ester production in nitrogen-rich conditions by metabolically engineered Acinetobacter baylyi ADP1

    doi: 10.1016/j.mec.2020.e00128

    Figure Lengend Snippet: (A) Growth (represented as OD 600 ) of the strains W1 and ADP1 WT in 10 and 20 ​g/L casein amino acids. (B) Comparison of WE content between W1 and ADP1 WT in the exponential phase when grown in 10 and 20 ​g/L casein amino acids. Cells of both strains were harvested during exponential phase; ADP1 WT was harvested after 4 ​h, and WI was harvested after 11 ​h. Comparison of (C) CDW and (D) WE content between W1 and ADP1 when cultivated with 20 ​g/L yeast extract. The results represent the mean of two replicates and the error bars represent the standard deviations. ∗∗ P ​

    Article Snippet: To construct the strain ADP1 ΔaceA , the cassette PT5 -kan r was cloned from previously contructed plasmid PT5 -kan r /pAK400c ( ) to the plasmid pUC57(ΔaceA ) containing the sequences flanking the gene aceA in the ADP1 genome (purchased from GenScript, USA) by digestion and ligation with the restriction enzymes AvrII and MfeI.

    Techniques:

    Comparison of (A) WE titer, (B) content and (C) CDW between ADP1 WT and W1 when grown in 50% baker’s yeast hydrolysate. The results represent the mean of two replicates and the error bars represent the standard deviations. (D) WE visualization by TLC. For each strain, the same amount of biomass was taken for lipid extraction. The extracted lipid was analyzed with TLC. Jojoba oil was used as the standard of WEs. ∗ P ​

    Journal: Metabolic Engineering Communications

    Article Title: Wax ester production in nitrogen-rich conditions by metabolically engineered Acinetobacter baylyi ADP1

    doi: 10.1016/j.mec.2020.e00128

    Figure Lengend Snippet: Comparison of (A) WE titer, (B) content and (C) CDW between ADP1 WT and W1 when grown in 50% baker’s yeast hydrolysate. The results represent the mean of two replicates and the error bars represent the standard deviations. (D) WE visualization by TLC. For each strain, the same amount of biomass was taken for lipid extraction. The extracted lipid was analyzed with TLC. Jojoba oil was used as the standard of WEs. ∗ P ​

    Article Snippet: To construct the strain ADP1 ΔaceA , the cassette PT5 -kan r was cloned from previously contructed plasmid PT5 -kan r /pAK400c ( ) to the plasmid pUC57(ΔaceA ) containing the sequences flanking the gene aceA in the ADP1 genome (purchased from GenScript, USA) by digestion and ligation with the restriction enzymes AvrII and MfeI.

    Techniques: Thin Layer Chromatography

    (A) Removal of the glyoxylate shunt hypothetically increases the availability of the central metabolite, acetyl-CoA, for fatty acid synthesis and energy generation. (B) The simplified metabolic pathway for the synthesis of wax esters from glucose and amino acids by A. baylyi ADP1. In the engineered strain, the gene aceA encoding for the isocitrate lyase, was deleted, blocking the glyoxylate shunt. The gene acr1 , encoding for the fatty acyl-CoA reductase, was overexpressed to facilitate wax ester production. In the final step of wax ester synthesis, fatty alcohol is esterified with fatty acyl-CoA by wax ester synthase (wax-dgaT).

    Journal: Metabolic Engineering Communications

    Article Title: Wax ester production in nitrogen-rich conditions by metabolically engineered Acinetobacter baylyi ADP1

    doi: 10.1016/j.mec.2020.e00128

    Figure Lengend Snippet: (A) Removal of the glyoxylate shunt hypothetically increases the availability of the central metabolite, acetyl-CoA, for fatty acid synthesis and energy generation. (B) The simplified metabolic pathway for the synthesis of wax esters from glucose and amino acids by A. baylyi ADP1. In the engineered strain, the gene aceA encoding for the isocitrate lyase, was deleted, blocking the glyoxylate shunt. The gene acr1 , encoding for the fatty acyl-CoA reductase, was overexpressed to facilitate wax ester production. In the final step of wax ester synthesis, fatty alcohol is esterified with fatty acyl-CoA by wax ester synthase (wax-dgaT).

    Article Snippet: To construct the strain ADP1 ΔaceA , the cassette PT5 -kan r was cloned from previously contructed plasmid PT5 -kan r /pAK400c ( ) to the plasmid pUC57(ΔaceA ) containing the sequences flanking the gene aceA in the ADP1 genome (purchased from GenScript, USA) by digestion and ligation with the restriction enzymes AvrII and MfeI.

    Techniques: Blocking Assay

    (A) Cumulative luminescence signal generated by W1+iluxAB, ADP1 Acr1+iluxAB, ADP1 Δ aceA ​+ ​iluxAB, ADP1+iluxAB and ADP1 FAR-neg.+iluxAB when grown in 200 ​mM glucose. All the strains express bacterial luciferase LuxAB that produces luminescence when reacting with the WE synthesis pathway intermediates, fatty aldehydes, indicating the relative activity of the synthesis pathway. The results represent the mean of two replicates and the error bars represent the standard deviations. (B) Visualization of the produced WEs by TLC (Thin-layer chromatography) analysis. The strains W1, ADP1 Acr1, ADP1 Δ aceA, and ADP1 WT were cultivated in 200 ​mM glucose for 48 ​h. For each strain, the same amount of biomass was taken for lipid extraction. Jojoba oil was used as the standard for WEs. Two replicates were analyzed and one representative image is shown.

    Journal: Metabolic Engineering Communications

    Article Title: Wax ester production in nitrogen-rich conditions by metabolically engineered Acinetobacter baylyi ADP1

    doi: 10.1016/j.mec.2020.e00128

    Figure Lengend Snippet: (A) Cumulative luminescence signal generated by W1+iluxAB, ADP1 Acr1+iluxAB, ADP1 Δ aceA ​+ ​iluxAB, ADP1+iluxAB and ADP1 FAR-neg.+iluxAB when grown in 200 ​mM glucose. All the strains express bacterial luciferase LuxAB that produces luminescence when reacting with the WE synthesis pathway intermediates, fatty aldehydes, indicating the relative activity of the synthesis pathway. The results represent the mean of two replicates and the error bars represent the standard deviations. (B) Visualization of the produced WEs by TLC (Thin-layer chromatography) analysis. The strains W1, ADP1 Acr1, ADP1 Δ aceA, and ADP1 WT were cultivated in 200 ​mM glucose for 48 ​h. For each strain, the same amount of biomass was taken for lipid extraction. Jojoba oil was used as the standard for WEs. Two replicates were analyzed and one representative image is shown.

    Article Snippet: To construct the strain ADP1 ΔaceA , the cassette PT5 -kan r was cloned from previously contructed plasmid PT5 -kan r /pAK400c ( ) to the plasmid pUC57(ΔaceA ) containing the sequences flanking the gene aceA in the ADP1 genome (purchased from GenScript, USA) by digestion and ligation with the restriction enzymes AvrII and MfeI.

    Techniques: Generated, Luciferase, Activity Assay, Produced, Thin Layer Chromatography

    (A) Growth (determined as OD 600 ) and (B) cumulative luminescence of W1+iluxAB, ADP1 Acr1+iluxAB, ADP1 Δ aceA ​+ ​iluxAB, and ADP1+iluxAB grown on alanine, asparagine, aspartate, glutamate, glutamine, and proline (the 6 amino acids can be used by A. baylyi ADP1 as sole carbon source). All the strains express bacterial luciferase LuxAB that produces luminescence when reacting with the WE synthesis pathway intermediates, fatty aldehydes, indicating the relative activity of the synthesis pathway. The results represent the mean of two replicates and the error bars represent the standard deviations.

    Journal: Metabolic Engineering Communications

    Article Title: Wax ester production in nitrogen-rich conditions by metabolically engineered Acinetobacter baylyi ADP1

    doi: 10.1016/j.mec.2020.e00128

    Figure Lengend Snippet: (A) Growth (determined as OD 600 ) and (B) cumulative luminescence of W1+iluxAB, ADP1 Acr1+iluxAB, ADP1 Δ aceA ​+ ​iluxAB, and ADP1+iluxAB grown on alanine, asparagine, aspartate, glutamate, glutamine, and proline (the 6 amino acids can be used by A. baylyi ADP1 as sole carbon source). All the strains express bacterial luciferase LuxAB that produces luminescence when reacting with the WE synthesis pathway intermediates, fatty aldehydes, indicating the relative activity of the synthesis pathway. The results represent the mean of two replicates and the error bars represent the standard deviations.

    Article Snippet: To construct the strain ADP1 ΔaceA , the cassette PT5 -kan r was cloned from previously contructed plasmid PT5 -kan r /pAK400c ( ) to the plasmid pUC57(ΔaceA ) containing the sequences flanking the gene aceA in the ADP1 genome (purchased from GenScript, USA) by digestion and ligation with the restriction enzymes AvrII and MfeI.

    Techniques: Luciferase, Activity Assay

    Comparison of (A) WE titer, (B) yield, (C) content and (D) CDW between ADP1 WT and W1 after 24 ​h and 48 ​h of cultivation with 200 ​mM glucose as carbon source. The results represent the mean of two replicates and the error bars represent the standard deviations. ∗ P ​

    Journal: Metabolic Engineering Communications

    Article Title: Wax ester production in nitrogen-rich conditions by metabolically engineered Acinetobacter baylyi ADP1

    doi: 10.1016/j.mec.2020.e00128

    Figure Lengend Snippet: Comparison of (A) WE titer, (B) yield, (C) content and (D) CDW between ADP1 WT and W1 after 24 ​h and 48 ​h of cultivation with 200 ​mM glucose as carbon source. The results represent the mean of two replicates and the error bars represent the standard deviations. ∗ P ​

    Article Snippet: To construct the strain ADP1 ΔaceA , the cassette PT5 -kan r was cloned from previously contructed plasmid PT5 -kan r /pAK400c ( ) to the plasmid pUC57(ΔaceA ) containing the sequences flanking the gene aceA in the ADP1 genome (purchased from GenScript, USA) by digestion and ligation with the restriction enzymes AvrII and MfeI.

    Techniques:

    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.

    Journal: Journal of Virology

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

    doi: 10.1128/JVI.00196-18

    Figure Lengend 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.

    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).

    Techniques: 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.

    Journal: Journal of Virology

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

    doi: 10.1128/JVI.00196-18

    Figure Lengend 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.

    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).

    Techniques: 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.

    Journal: Journal of Virology

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

    doi: 10.1128/JVI.00196-18

    Figure Lengend 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.

    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).

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