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pnl4 3  (New England Biolabs)


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    Structured Review

    New England Biolabs pnl4 3
    Pnl4 3, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 9775 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/pnl4+3/Q5+Site-Directed+Mutagenesis+Kit/10__3390_slash_microorganisms14051102-52-8-15
    Average 99 stars, based on 9775 article reviews
    pnl4 3 - by Bioz Stars, 2026-09
    99/100 stars

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    Related Articles

    Generated:

    Article Title: Macropinosomes are a site of HIV-1 entry into primary CD4 + T cells
    Article Snippet: .. A replication-competent HIV-1 molecular clone encoding NanoLuc, pNL-NI, was generated by the restriction enzyme digestion of pCMV6-AC/partial NLenv-NanoLuc-IRES-nef and pNL4-3 at BamHI and XhoI sites and the ligation of these digested plasmids. pCMV6-AC/partial NLenv-nef was generated by the restriction enzyme digestion of pCMV6-AC (Origene) and pNL4-3 at BamHI and XhoI sites and the ligation of these digested plasmids. pCMV6-AC/partial NLenv-NanoLuc-IRES-nef was generated by introducing nanoluc and the IRES sequence between env and nef ORFs by using Gibson Assembly Kit (NEB). ..

    Article Title: Macropinosomes are a site of HIV-1 entry into primary CD4 + T cells
    Article Snippet: .. A replication-competent HIV-1 molecular clone encoding NanoLuc, pNL-NI, was generated by the restriction enzyme digestion of pCMV6-AC/partial NLenv-NanoLuc-IRES-nef and pNL4-3 at BamHI and XhoI sites and the ligation of these digested plasmids. pCMV6-AC/partial NLenv-nef was generated by the restriction enzyme digestion of pCMV6-AC (Origene) and pNL4-3 at BamHI and XhoI sites and the ligation of these digested plasmids. pCMV6-AC/partial NLenvNanoLuc-IRES-nef was generated by introducing nanoluc and the IRES sequence between env and nef ORFs by using Gibson Assembly Kit (NEB). ..

    Ligation:

    Article Title: Macropinosomes are a site of HIV-1 entry into primary CD4 + T cells
    Article Snippet: .. A replication-competent HIV-1 molecular clone encoding NanoLuc, pNL-NI, was generated by the restriction enzyme digestion of pCMV6-AC/partial NLenv-NanoLuc-IRES-nef and pNL4-3 at BamHI and XhoI sites and the ligation of these digested plasmids. pCMV6-AC/partial NLenv-nef was generated by the restriction enzyme digestion of pCMV6-AC (Origene) and pNL4-3 at BamHI and XhoI sites and the ligation of these digested plasmids. pCMV6-AC/partial NLenv-NanoLuc-IRES-nef was generated by introducing nanoluc and the IRES sequence between env and nef ORFs by using Gibson Assembly Kit (NEB). ..

    Article Title: Macropinosomes are a site of HIV-1 entry into primary CD4 + T cells
    Article Snippet: .. A replication-competent HIV-1 molecular clone encoding NanoLuc, pNL-NI, was generated by the restriction enzyme digestion of pCMV6-AC/partial NLenv-NanoLuc-IRES-nef and pNL4-3 at BamHI and XhoI sites and the ligation of these digested plasmids. pCMV6-AC/partial NLenv-nef was generated by the restriction enzyme digestion of pCMV6-AC (Origene) and pNL4-3 at BamHI and XhoI sites and the ligation of these digested plasmids. pCMV6-AC/partial NLenvNanoLuc-IRES-nef was generated by introducing nanoluc and the IRES sequence between env and nef ORFs by using Gibson Assembly Kit (NEB). ..

    Sequencing:

    Article Title: Macropinosomes are a site of HIV-1 entry into primary CD4 + T cells
    Article Snippet: .. A replication-competent HIV-1 molecular clone encoding NanoLuc, pNL-NI, was generated by the restriction enzyme digestion of pCMV6-AC/partial NLenv-NanoLuc-IRES-nef and pNL4-3 at BamHI and XhoI sites and the ligation of these digested plasmids. pCMV6-AC/partial NLenv-nef was generated by the restriction enzyme digestion of pCMV6-AC (Origene) and pNL4-3 at BamHI and XhoI sites and the ligation of these digested plasmids. pCMV6-AC/partial NLenv-NanoLuc-IRES-nef was generated by introducing nanoluc and the IRES sequence between env and nef ORFs by using Gibson Assembly Kit (NEB). ..

    Article Title: Macropinosomes are a site of HIV-1 entry into primary CD4 + T cells
    Article Snippet: .. A replication-competent HIV-1 molecular clone encoding NanoLuc, pNL-NI, was generated by the restriction enzyme digestion of pCMV6-AC/partial NLenv-NanoLuc-IRES-nef and pNL4-3 at BamHI and XhoI sites and the ligation of these digested plasmids. pCMV6-AC/partial NLenv-nef was generated by the restriction enzyme digestion of pCMV6-AC (Origene) and pNL4-3 at BamHI and XhoI sites and the ligation of these digested plasmids. pCMV6-AC/partial NLenvNanoLuc-IRES-nef was generated by introducing nanoluc and the IRES sequence between env and nef ORFs by using Gibson Assembly Kit (NEB). ..

    Mutagenesis:

    Article Title: GigaAssay - An adaptable high-throughput saturation mutagenesis assay platform.
    Article Snippet: The plasmid pLjm1_mcs was made by introducing compatible EcoRI, SalI, and AsiSI restriction enzyme sites in the pLjm1-Empty (Addgene) vector for cloning of the Tat variant library. .. Tat or mutant Tat encoding a C27S mutation was PCR amplified from pNL4–3 as a template with Q5® High-Fidelity DNA Polymerase (New England Biolabs) and cloned into EcoRI/SalI digested pLjm1_mcs1. ..

    Article Title: Mutation-Tolerant Inhibition of HIV-1 Integrase Strand Transfer by Secondary Metabolites from the Endophytic Fungus Alternaria alternata PO4PR2
    Article Snippet: Mutagenic primers (Table 2) were designed using the NEBaseChanger primer design tool (New England Biolabs, MA, USA). .. Mutations were introduced into the HIV-1 molecular clone pNL4.3 using the Q5 site-directed mutagenesis kit (New England Biolabs, Ipswich, MA, USA). ..

    Article Title: GigaAssay – An adaptable high-throughput saturation mutagenesis assay platform
    Article Snippet: The plasmid pLjm1_mcs was made by introducing compatible Eco RI, Sal I, and Asi SI restriction enzyme sites in the pLjm1-Empty (Addgene) vector for cloning of the Tat variant library. .. Tat or mutant Tat encoding a C27S mutation was PCR amplified from pNL4–3 as a template with Q5® High-Fidelity DNA Polymerase (New England Biolabs) and cloned into EcoRI/SalI digested pLjm1_mcs1. ..

    Article Title: GigaAssay – a high-throughput assay system for molecular functions and cell processes
    Article Snippet: The plasmid pLjm1_mcs was made by introducing compatible EcoRI, SalI, and AsiSI restriction enzyme sites in the pLjm1-Empty (Addgene) vector for cloning of the Tat variant library. .. Tat or mutant Tat encoding a C27S mutation was PCR amplified from pNL4-3 as a template with Q5® High-Fidelity DNA Polymerase (New England Biolabs) and cloned into EcoRI/SalI digested pLjm1_mcs1. ..

    Polymerase Chain Reaction:

    Article Title: GigaAssay - An adaptable high-throughput saturation mutagenesis assay platform.
    Article Snippet: The plasmid pLjm1_mcs was made by introducing compatible EcoRI, SalI, and AsiSI restriction enzyme sites in the pLjm1-Empty (Addgene) vector for cloning of the Tat variant library. .. Tat or mutant Tat encoding a C27S mutation was PCR amplified from pNL4–3 as a template with Q5® High-Fidelity DNA Polymerase (New England Biolabs) and cloned into EcoRI/SalI digested pLjm1_mcs1. ..

    Article Title: Cytosine deamination and the precipitous decline of spontaneous mutation during Earth's history
    Article Snippet: .. This region was PCR amplified from a convenient plasmid subclone of pNL4-3 and cloned into pBlueScript+ spanning from the EcoRI site to the HindIII site using the Gibson Assembly method (New England BioLabs). ..

    Article Title: GigaAssay – An adaptable high-throughput saturation mutagenesis assay platform
    Article Snippet: The plasmid pLjm1_mcs was made by introducing compatible Eco RI, Sal I, and Asi SI restriction enzyme sites in the pLjm1-Empty (Addgene) vector for cloning of the Tat variant library. .. Tat or mutant Tat encoding a C27S mutation was PCR amplified from pNL4–3 as a template with Q5® High-Fidelity DNA Polymerase (New England Biolabs) and cloned into EcoRI/SalI digested pLjm1_mcs1. ..

    Article Title: GigaAssay – a high-throughput assay system for molecular functions and cell processes
    Article Snippet: The plasmid pLjm1_mcs was made by introducing compatible EcoRI, SalI, and AsiSI restriction enzyme sites in the pLjm1-Empty (Addgene) vector for cloning of the Tat variant library. .. Tat or mutant Tat encoding a C27S mutation was PCR amplified from pNL4-3 as a template with Q5® High-Fidelity DNA Polymerase (New England Biolabs) and cloned into EcoRI/SalI digested pLjm1_mcs1. ..

    Amplification:

    Article Title: GigaAssay - An adaptable high-throughput saturation mutagenesis assay platform.
    Article Snippet: The plasmid pLjm1_mcs was made by introducing compatible EcoRI, SalI, and AsiSI restriction enzyme sites in the pLjm1-Empty (Addgene) vector for cloning of the Tat variant library. .. Tat or mutant Tat encoding a C27S mutation was PCR amplified from pNL4–3 as a template with Q5® High-Fidelity DNA Polymerase (New England Biolabs) and cloned into EcoRI/SalI digested pLjm1_mcs1. ..

    Article Title: Cytosine deamination and the precipitous decline of spontaneous mutation during Earth's history
    Article Snippet: .. This region was PCR amplified from a convenient plasmid subclone of pNL4-3 and cloned into pBlueScript+ spanning from the EcoRI site to the HindIII site using the Gibson Assembly method (New England BioLabs). ..

    Article Title: GigaAssay – An adaptable high-throughput saturation mutagenesis assay platform
    Article Snippet: The plasmid pLjm1_mcs was made by introducing compatible Eco RI, Sal I, and Asi SI restriction enzyme sites in the pLjm1-Empty (Addgene) vector for cloning of the Tat variant library. .. Tat or mutant Tat encoding a C27S mutation was PCR amplified from pNL4–3 as a template with Q5® High-Fidelity DNA Polymerase (New England Biolabs) and cloned into EcoRI/SalI digested pLjm1_mcs1. ..

    Article Title: GigaAssay – a high-throughput assay system for molecular functions and cell processes
    Article Snippet: The plasmid pLjm1_mcs was made by introducing compatible EcoRI, SalI, and AsiSI restriction enzyme sites in the pLjm1-Empty (Addgene) vector for cloning of the Tat variant library. .. Tat or mutant Tat encoding a C27S mutation was PCR amplified from pNL4-3 as a template with Q5® High-Fidelity DNA Polymerase (New England Biolabs) and cloned into EcoRI/SalI digested pLjm1_mcs1. ..

    Clone Assay:

    Article Title: GigaAssay - An adaptable high-throughput saturation mutagenesis assay platform.
    Article Snippet: The plasmid pLjm1_mcs was made by introducing compatible EcoRI, SalI, and AsiSI restriction enzyme sites in the pLjm1-Empty (Addgene) vector for cloning of the Tat variant library. .. Tat or mutant Tat encoding a C27S mutation was PCR amplified from pNL4–3 as a template with Q5® High-Fidelity DNA Polymerase (New England Biolabs) and cloned into EcoRI/SalI digested pLjm1_mcs1. ..

    Article Title: Cytosine deamination and the precipitous decline of spontaneous mutation during Earth's history
    Article Snippet: .. This region was PCR amplified from a convenient plasmid subclone of pNL4-3 and cloned into pBlueScript+ spanning from the EcoRI site to the HindIII site using the Gibson Assembly method (New England BioLabs). ..

    Article Title: GigaAssay – An adaptable high-throughput saturation mutagenesis assay platform
    Article Snippet: The plasmid pLjm1_mcs was made by introducing compatible Eco RI, Sal I, and Asi SI restriction enzyme sites in the pLjm1-Empty (Addgene) vector for cloning of the Tat variant library. .. Tat or mutant Tat encoding a C27S mutation was PCR amplified from pNL4–3 as a template with Q5® High-Fidelity DNA Polymerase (New England Biolabs) and cloned into EcoRI/SalI digested pLjm1_mcs1. ..

    Article Title: GigaAssay – a high-throughput assay system for molecular functions and cell processes
    Article Snippet: The plasmid pLjm1_mcs was made by introducing compatible EcoRI, SalI, and AsiSI restriction enzyme sites in the pLjm1-Empty (Addgene) vector for cloning of the Tat variant library. .. Tat or mutant Tat encoding a C27S mutation was PCR amplified from pNL4-3 as a template with Q5® High-Fidelity DNA Polymerase (New England Biolabs) and cloned into EcoRI/SalI digested pLjm1_mcs1. ..

    Plasmid Preparation:

    Article Title: Cytosine deamination and the precipitous decline of spontaneous mutation during Earth's history
    Article Snippet: .. This region was PCR amplified from a convenient plasmid subclone of pNL4-3 and cloned into pBlueScript+ spanning from the EcoRI site to the HindIII site using the Gibson Assembly method (New England BioLabs). ..

    Article Title: The Use of Minimal RNA Toeholds to Trigger the Activation of Multiple Functionalities
    Article Snippet: HeLa cells were cultured in a 24-well plate, in a 5% CO 2 incubator, in antibiotic-free DMEM (Gibco BRL) supplemented with 5% FBS (Atlanta biologicals). .. Nucleic acids and L2K were mixed an concentrations higher than recommended by the manufacturer; for each well, 0.66 μ g HIV-1 infectious clone pNL4–3, 49 0.05 μ g Gaussia luciferase reporter plasmid pGLuc (NEB) and either 2.5 or 10 pmol of RNA/DNA hybrids, were combined in 15 μ L of Optimem. ..

    Luciferase:

    Article Title: The Use of Minimal RNA Toeholds to Trigger the Activation of Multiple Functionalities
    Article Snippet: HeLa cells were cultured in a 24-well plate, in a 5% CO 2 incubator, in antibiotic-free DMEM (Gibco BRL) supplemented with 5% FBS (Atlanta biologicals). .. Nucleic acids and L2K were mixed an concentrations higher than recommended by the manufacturer; for each well, 0.66 μ g HIV-1 infectious clone pNL4–3, 49 0.05 μ g Gaussia luciferase reporter plasmid pGLuc (NEB) and either 2.5 or 10 pmol of RNA/DNA hybrids, were combined in 15 μ L of Optimem. ..



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    (A) Western blot showing expression of free and conjugated forms of 3xFLAG-SUMO1 Q92R and 3xFLAG-SUMO2 Q88R in HeLa cells. Expression of endogenous wild-type SUMO is shown for comparison. “-” = Untransfected Control. (B) SUMO-modified RanGAP1 is detected following immunoprecipitation with a FLAG or HIS antibody in cells expressing 3xFLAG-SUMO1 Q92R or HIS 6 -SUMO1 Q92R , respectively. Whole cell lysate (WCL) was used as input for IPs. Non-immune IgG = IP control. (C) Expression of exogenous SUMO does not interfere with HIV-1 infection. Cell lines were infected with equal volumes of viral supernatants containing VSV-G pseudotyped HIV-1 <t>pNL4-3</t> <t>deltaENV-EGFP</t> reporter virus. %GFP+ cells and GFP mean fluorescence intensity (MFI) was assessed by flow cytometry at 24 hpi. “-” = Untransfected Control.
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    Image Search Results


    (A) Western blot showing expression of free and conjugated forms of 3xFLAG-SUMO1 Q92R and 3xFLAG-SUMO2 Q88R in HeLa cells. Expression of endogenous wild-type SUMO is shown for comparison. “-” = Untransfected Control. (B) SUMO-modified RanGAP1 is detected following immunoprecipitation with a FLAG or HIS antibody in cells expressing 3xFLAG-SUMO1 Q92R or HIS 6 -SUMO1 Q92R , respectively. Whole cell lysate (WCL) was used as input for IPs. Non-immune IgG = IP control. (C) Expression of exogenous SUMO does not interfere with HIV-1 infection. Cell lines were infected with equal volumes of viral supernatants containing VSV-G pseudotyped HIV-1 pNL4-3 deltaENV-EGFP reporter virus. %GFP+ cells and GFP mean fluorescence intensity (MFI) was assessed by flow cytometry at 24 hpi. “-” = Untransfected Control.

    Journal: bioRxiv

    Article Title: A proteome-wide, MS-based screen identifies SUMOylation of host RNA splicing factors induced by HIV-1 infection

    doi: 10.1101/2025.03.26.645526

    Figure Lengend Snippet: (A) Western blot showing expression of free and conjugated forms of 3xFLAG-SUMO1 Q92R and 3xFLAG-SUMO2 Q88R in HeLa cells. Expression of endogenous wild-type SUMO is shown for comparison. “-” = Untransfected Control. (B) SUMO-modified RanGAP1 is detected following immunoprecipitation with a FLAG or HIS antibody in cells expressing 3xFLAG-SUMO1 Q92R or HIS 6 -SUMO1 Q92R , respectively. Whole cell lysate (WCL) was used as input for IPs. Non-immune IgG = IP control. (C) Expression of exogenous SUMO does not interfere with HIV-1 infection. Cell lines were infected with equal volumes of viral supernatants containing VSV-G pseudotyped HIV-1 pNL4-3 deltaENV-EGFP reporter virus. %GFP+ cells and GFP mean fluorescence intensity (MFI) was assessed by flow cytometry at 24 hpi. “-” = Untransfected Control.

    Article Snippet: Cells were co-transfected with 15 µg of the pNL4-3 deltaENV-EGFP reporter (Centre for AIDS reagents #100616) and 5 µg of the pCMV-VSV-G vector (Addgene #8454) using Lipofectamine 3000 (Invitrogen #L3000075, per manufacturer’s protocol) in serum-free DMEM.

    Techniques: Western Blot, Expressing, Comparison, Control, Modification, Immunoprecipitation, Infection, Virus, Fluorescence, Flow Cytometry

    (A) Western blot showing the level of SUMO1-modified and unmodified RanGAP1 in HeLa cells treated with the SUMOylation inhibitor TAK-981 versus the DMSO control (“-”). (B) Western blot showing the level of SUMO1-conjugated A2B1 and A3 in HIV-1-infected cells treated with or without TAK-981. Uninfected, untreated cells were used as a control. Crude lysate from the corresponding conditions was used as input for IPs (5% of total protein loaded in IPs). For both A and B, TAK-981 was used at a concentration of 12 µM. DMSO = untreated control. (C) Western blot analysis of SUMO1-modified A2B1 and A3 in cells infected with Env mutant HIV-1 (pNL4-3 deltaENV-EGFP) lacking the VSV-G envelope (“-VSV-G HIV”) versus the uninfected control. Cells were harvested at 48 hpi. (D) Western blot showing the level of SUMO-modified A2B1 or A3 in HIV-1-infected HeLa cells versus uninfected controls at 6, 12, 24, and 48 hpi. (E, F) Western blot analysis of SUMO1-modified A2B1 and A3 in HIV-1-infected Jurkat cells (E) or K562 cells (F) versus uninfected controls at 48 hpi. (G) Western blot analysis of SUMO1-modified A2B1 and A3 in MMLV-infected HeLa cells versus uninfected controls. Cells were infected with VSV-G pseudotyped MMLV (pNCA-GFP) and harvested at 48 hpi for lysate preparation. Crude lysate input for each condition is shown for comparison. For all, SUMO1-conjugated proteins were immunoprecipitated using anti-SUMO1 antibodies and samples were analyzed by western blot for A2B1 and A3. Arrows indicate SUMO1-modified proteins. Stars indicate unmodified proteins. For C-G, non-immune IgG = IP control and SUMO1-modified RanGAP1 = loading control for IPs.

    Journal: bioRxiv

    Article Title: A proteome-wide, MS-based screen identifies SUMOylation of host RNA splicing factors induced by HIV-1 infection

    doi: 10.1101/2025.03.26.645526

    Figure Lengend Snippet: (A) Western blot showing the level of SUMO1-modified and unmodified RanGAP1 in HeLa cells treated with the SUMOylation inhibitor TAK-981 versus the DMSO control (“-”). (B) Western blot showing the level of SUMO1-conjugated A2B1 and A3 in HIV-1-infected cells treated with or without TAK-981. Uninfected, untreated cells were used as a control. Crude lysate from the corresponding conditions was used as input for IPs (5% of total protein loaded in IPs). For both A and B, TAK-981 was used at a concentration of 12 µM. DMSO = untreated control. (C) Western blot analysis of SUMO1-modified A2B1 and A3 in cells infected with Env mutant HIV-1 (pNL4-3 deltaENV-EGFP) lacking the VSV-G envelope (“-VSV-G HIV”) versus the uninfected control. Cells were harvested at 48 hpi. (D) Western blot showing the level of SUMO-modified A2B1 or A3 in HIV-1-infected HeLa cells versus uninfected controls at 6, 12, 24, and 48 hpi. (E, F) Western blot analysis of SUMO1-modified A2B1 and A3 in HIV-1-infected Jurkat cells (E) or K562 cells (F) versus uninfected controls at 48 hpi. (G) Western blot analysis of SUMO1-modified A2B1 and A3 in MMLV-infected HeLa cells versus uninfected controls. Cells were infected with VSV-G pseudotyped MMLV (pNCA-GFP) and harvested at 48 hpi for lysate preparation. Crude lysate input for each condition is shown for comparison. For all, SUMO1-conjugated proteins were immunoprecipitated using anti-SUMO1 antibodies and samples were analyzed by western blot for A2B1 and A3. Arrows indicate SUMO1-modified proteins. Stars indicate unmodified proteins. For C-G, non-immune IgG = IP control and SUMO1-modified RanGAP1 = loading control for IPs.

    Article Snippet: Cells were co-transfected with 15 µg of the pNL4-3 deltaENV-EGFP reporter (Centre for AIDS reagents #100616) and 5 µg of the pCMV-VSV-G vector (Addgene #8454) using Lipofectamine 3000 (Invitrogen #L3000075, per manufacturer’s protocol) in serum-free DMEM.

    Techniques: Western Blot, Modification, Control, Infection, Concentration Assay, Mutagenesis, Comparison, Immunoprecipitation