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Establishment of reporter T cells and CRISPR/Cas9-mediated knock-in of a candidate T cell receptor (TCR). ( A ) Surface marker expression of parental Jurkat E6-1 cells. Histograms show the expression levels of the indicated markers. ( B ) Surface marker expression of engineered reporter T cells. Histograms show the expression levels of the indicated markers. ( C ) Schematic overview of the genetic modifications performed to generate reporter T cells. Parental Jurkat E6-1 cells were subjected to CRISPR/Cas9-mediated knockout of CD4, FAS, and TCRα, and <t>lentiviral</t> transduction of CD8α, CD8β, Cas9, and the NFAT–Luc–P2A–eGFP reporter cassette. ( D ) Targeted reconstitution of the TCR in reporter T cells at the endogenous TCRβ locus. ( E ) Flow cytometric analysis of CD3 restoration in reporter T cells after targeted reconstitution with the NY-ESO-1–specific 1G4 TCR. CD3 + cells were sorted to generate a uniform population of TCR-expressing reporter T cells. Pseudocolor plots indicate event density (blue, lower; red, higher).
Lentiviral Nfat Luciferase Egfp Reporter Construct, supplied by BPS Bioscience, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Establishment of reporter T cells and CRISPR/Cas9-mediated knock-in of a candidate T cell receptor (TCR). ( A ) Surface marker expression of parental Jurkat E6-1 cells. Histograms show the expression levels of the indicated markers. ( B ) Surface marker expression of engineered reporter T cells. Histograms show the expression levels of the indicated markers. ( C ) Schematic overview of the genetic modifications performed to generate reporter T cells. Parental Jurkat E6-1 cells were subjected to CRISPR/Cas9-mediated knockout of CD4, FAS, and TCRα, and <t>lentiviral</t> transduction of CD8α, CD8β, Cas9, and the NFAT–Luc–P2A–eGFP reporter cassette. ( D ) Targeted reconstitution of the TCR in reporter T cells at the endogenous TCRβ locus. ( E ) Flow cytometric analysis of CD3 restoration in reporter T cells after targeted reconstitution with the NY-ESO-1–specific 1G4 TCR. CD3 + cells were sorted to generate a uniform population of TCR-expressing reporter T cells. Pseudocolor plots indicate event density (blue, lower; red, higher).
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Establishment of reporter T cells and CRISPR/Cas9-mediated knock-in of a candidate T cell receptor (TCR). ( A ) Surface marker expression of parental Jurkat E6-1 cells. Histograms show the expression levels of the indicated markers. ( B ) Surface marker expression of engineered reporter T cells. Histograms show the expression levels of the indicated markers. ( C ) Schematic overview of the genetic modifications performed to generate reporter T cells. Parental Jurkat E6-1 cells were subjected to CRISPR/Cas9-mediated knockout of CD4, FAS, and TCRα, and <t>lentiviral</t> transduction of CD8α, CD8β, Cas9, and the NFAT–Luc–P2A–eGFP reporter cassette. ( D ) Targeted reconstitution of the TCR in reporter T cells at the endogenous TCRβ locus. ( E ) Flow cytometric analysis of CD3 restoration in reporter T cells after targeted reconstitution with the NY-ESO-1–specific 1G4 TCR. CD3 + cells were sorted to generate a uniform population of TCR-expressing reporter T cells. Pseudocolor plots indicate event density (blue, lower; red, higher).
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Establishment of reporter T cells and CRISPR/Cas9-mediated knock-in of a candidate T cell receptor (TCR). ( A ) Surface marker expression of parental Jurkat E6-1 cells. Histograms show the expression levels of the indicated markers. ( B ) Surface marker expression of engineered reporter T cells. Histograms show the expression levels of the indicated markers. ( C ) Schematic overview of the genetic modifications performed to generate reporter T cells. Parental Jurkat E6-1 cells were subjected to CRISPR/Cas9-mediated knockout of CD4, FAS, and TCRα, and <t>lentiviral</t> transduction of CD8α, CD8β, Cas9, and the NFAT–Luc–P2A–eGFP reporter cassette. ( D ) Targeted reconstitution of the TCR in reporter T cells at the endogenous TCRβ locus. ( E ) Flow cytometric analysis of CD3 restoration in reporter T cells after targeted reconstitution with the NY-ESO-1–specific 1G4 TCR. CD3 + cells were sorted to generate a uniform population of TCR-expressing reporter T cells. Pseudocolor plots indicate event density (blue, lower; red, higher).
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Establishment of reporter T cells and CRISPR/Cas9-mediated knock-in of a candidate T cell receptor (TCR). ( A ) Surface marker expression of parental Jurkat E6-1 cells. Histograms show the expression levels of the indicated markers. ( B ) Surface marker expression of engineered reporter T cells. Histograms show the expression levels of the indicated markers. ( C ) Schematic overview of the genetic modifications performed to generate reporter T cells. Parental Jurkat E6-1 cells were subjected to CRISPR/Cas9-mediated knockout of CD4, FAS, and TCRα, and <t>lentiviral</t> transduction of CD8α, CD8β, Cas9, and the NFAT–Luc–P2A–eGFP reporter cassette. ( D ) Targeted reconstitution of the TCR in reporter T cells at the endogenous TCRβ locus. ( E ) Flow cytometric analysis of CD3 restoration in reporter T cells after targeted reconstitution with the NY-ESO-1–specific 1G4 TCR. CD3 + cells were sorted to generate a uniform population of TCR-expressing reporter T cells. Pseudocolor plots indicate event density (blue, lower; red, higher).
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Establishment of reporter T cells and CRISPR/Cas9-mediated knock-in of a candidate T cell receptor (TCR). ( A ) Surface marker expression of parental Jurkat E6-1 cells. Histograms show the expression levels of the indicated markers. ( B ) Surface marker expression of engineered reporter T cells. Histograms show the expression levels of the indicated markers. ( C ) Schematic overview of the genetic modifications performed to generate reporter T cells. Parental Jurkat E6-1 cells were subjected to CRISPR/Cas9-mediated knockout of CD4, FAS, and TCRα, and <t>lentiviral</t> transduction of CD8α, CD8β, Cas9, and the NFAT–Luc–P2A–eGFP reporter cassette. ( D ) Targeted reconstitution of the TCR in reporter T cells at the endogenous TCRβ locus. ( E ) Flow cytometric analysis of CD3 restoration in reporter T cells after targeted reconstitution with the NY-ESO-1–specific 1G4 TCR. CD3 + cells were sorted to generate a uniform population of TCR-expressing reporter T cells. Pseudocolor plots indicate event density (blue, lower; red, higher).
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Establishment of reporter T cells and CRISPR/Cas9-mediated knock-in of a candidate T cell receptor (TCR). ( A ) Surface marker expression of parental Jurkat E6-1 cells. Histograms show the expression levels of the indicated markers. ( B ) Surface marker expression of engineered reporter T cells. Histograms show the expression levels of the indicated markers. ( C ) Schematic overview of the genetic modifications performed to generate reporter T cells. Parental Jurkat E6-1 cells were subjected to CRISPR/Cas9-mediated knockout of CD4, FAS, and TCRα, and <t>lentiviral</t> transduction of CD8α, CD8β, Cas9, and the NFAT–Luc–P2A–eGFP reporter cassette. ( D ) Targeted reconstitution of the TCR in reporter T cells at the endogenous TCRβ locus. ( E ) Flow cytometric analysis of CD3 restoration in reporter T cells after targeted reconstitution with the NY-ESO-1–specific 1G4 TCR. CD3 + cells were sorted to generate a uniform population of TCR-expressing reporter T cells. Pseudocolor plots indicate event density (blue, lower; red, higher).
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Establishment of reporter T cells and CRISPR/Cas9-mediated knock-in of a candidate T cell receptor (TCR). ( A ) Surface marker expression of parental Jurkat E6-1 cells. Histograms show the expression levels of the indicated markers. ( B ) Surface marker expression of engineered reporter T cells. Histograms show the expression levels of the indicated markers. ( C ) Schematic overview of the genetic modifications performed to generate reporter T cells. Parental Jurkat E6-1 cells were subjected to CRISPR/Cas9-mediated knockout of CD4, FAS, and TCRα, and <t>lentiviral</t> transduction of CD8α, CD8β, Cas9, and the NFAT–Luc–P2A–eGFP reporter cassette. ( D ) Targeted reconstitution of the TCR in reporter T cells at the endogenous TCRβ locus. ( E ) Flow cytometric analysis of CD3 restoration in reporter T cells after targeted reconstitution with the NY-ESO-1–specific 1G4 TCR. CD3 + cells were sorted to generate a uniform population of TCR-expressing reporter T cells. Pseudocolor plots indicate event density (blue, lower; red, higher).
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A. Immunoblotting after expression of GFP-FBXO42, HA-ubiquitin and either Flag-PP4R2 or Flag-PP4C in HEK293T as indicated. Flag-PP4R2 or Flag-PP4C was isolated via Flag agarose beads pulldown before immunoblotting. Input samples are in the right panel. B. Immunoblotting after isolation of endogenous ubiquitinated proteins using recombinant GST-tagged UBA domain of UBIQLN protein from H4 parental cells or cells FBXO42 K/O. Input samples before immunoprecipitation are indicated – bottom panel . C. Schematic representation of the E-Stub proximity ubiquitin labelling and enrichment. BirA-FBXO42 was co-expressed with a biotin acceptor ubiquitin mutant (bio <t>GEF</t> <t>UBnc)</t> followed by biotin treatment. Nascent ubiquitinated substrates are thus biotinylated and immunoprecipitated. D. Immunoblotting pf PP4 complex after expression of (doxycycline-inducible) BirA– FBXO42 and biotin bio GEF UBnc-Ub in T-REx™ Hek293 cells post. Proteins were induced by doxycycline for 24 hours and supplemented with biotin for 3 hours with or without MG132/MLN4924 as indicated, before protein lysis and immunoprecipitation. Input samples indicated in top panel.
Lentivirus Expression Construct Tripz Bio Gef Ubnc Puro, supplied by Addgene inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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lentivirus stably expressing the circkiaa1797-silencing construct  (Shanghai Genechem Ltd)
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Shanghai Genechem Ltd lentivirus stably expressing the circkiaa1797-silencing construct
A. Immunoblotting after expression of GFP-FBXO42, HA-ubiquitin and either Flag-PP4R2 or Flag-PP4C in HEK293T as indicated. Flag-PP4R2 or Flag-PP4C was isolated via Flag agarose beads pulldown before immunoblotting. Input samples are in the right panel. B. Immunoblotting after isolation of endogenous ubiquitinated proteins using recombinant GST-tagged UBA domain of UBIQLN protein from H4 parental cells or cells FBXO42 K/O. Input samples before immunoprecipitation are indicated – bottom panel . C. Schematic representation of the E-Stub proximity ubiquitin labelling and enrichment. BirA-FBXO42 was co-expressed with a biotin acceptor ubiquitin mutant (bio <t>GEF</t> <t>UBnc)</t> followed by biotin treatment. Nascent ubiquitinated substrates are thus biotinylated and immunoprecipitated. D. Immunoblotting pf PP4 complex after expression of (doxycycline-inducible) BirA– FBXO42 and biotin bio GEF UBnc-Ub in T-REx™ Hek293 cells post. Proteins were induced by doxycycline for 24 hours and supplemented with biotin for 3 hours with or without MG132/MLN4924 as indicated, before protein lysis and immunoprecipitation. Input samples indicated in top panel.
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Image Search Results


Establishment of reporter T cells and CRISPR/Cas9-mediated knock-in of a candidate T cell receptor (TCR). ( A ) Surface marker expression of parental Jurkat E6-1 cells. Histograms show the expression levels of the indicated markers. ( B ) Surface marker expression of engineered reporter T cells. Histograms show the expression levels of the indicated markers. ( C ) Schematic overview of the genetic modifications performed to generate reporter T cells. Parental Jurkat E6-1 cells were subjected to CRISPR/Cas9-mediated knockout of CD4, FAS, and TCRα, and lentiviral transduction of CD8α, CD8β, Cas9, and the NFAT–Luc–P2A–eGFP reporter cassette. ( D ) Targeted reconstitution of the TCR in reporter T cells at the endogenous TCRβ locus. ( E ) Flow cytometric analysis of CD3 restoration in reporter T cells after targeted reconstitution with the NY-ESO-1–specific 1G4 TCR. CD3 + cells were sorted to generate a uniform population of TCR-expressing reporter T cells. Pseudocolor plots indicate event density (blue, lower; red, higher).

Journal: Cells

Article Title: NeoPAIR-T: Functional Mapping of Neoantigen–TCR Pairs Using a CRISPR-Engineered Jurkat Reporter System

doi: 10.3390/cells14221789

Figure Lengend Snippet: Establishment of reporter T cells and CRISPR/Cas9-mediated knock-in of a candidate T cell receptor (TCR). ( A ) Surface marker expression of parental Jurkat E6-1 cells. Histograms show the expression levels of the indicated markers. ( B ) Surface marker expression of engineered reporter T cells. Histograms show the expression levels of the indicated markers. ( C ) Schematic overview of the genetic modifications performed to generate reporter T cells. Parental Jurkat E6-1 cells were subjected to CRISPR/Cas9-mediated knockout of CD4, FAS, and TCRα, and lentiviral transduction of CD8α, CD8β, Cas9, and the NFAT–Luc–P2A–eGFP reporter cassette. ( D ) Targeted reconstitution of the TCR in reporter T cells at the endogenous TCRβ locus. ( E ) Flow cytometric analysis of CD3 restoration in reporter T cells after targeted reconstitution with the NY-ESO-1–specific 1G4 TCR. CD3 + cells were sorted to generate a uniform population of TCR-expressing reporter T cells. Pseudocolor plots indicate event density (blue, lower; red, higher).

Article Snippet: Finally, these cells were transduced with a lentiviral NFAT-Luciferase-eGFP reporter construct ( , BPS Bioscience, San Diego, CA, USA) at MOI of 20 and selected with 2 mg/mL G418 (FujiFilm Wako Pure Chemical Corporation).

Techniques: CRISPR, Knock-In, Marker, Expressing, Knock-Out, Transduction

A. Immunoblotting after expression of GFP-FBXO42, HA-ubiquitin and either Flag-PP4R2 or Flag-PP4C in HEK293T as indicated. Flag-PP4R2 or Flag-PP4C was isolated via Flag agarose beads pulldown before immunoblotting. Input samples are in the right panel. B. Immunoblotting after isolation of endogenous ubiquitinated proteins using recombinant GST-tagged UBA domain of UBIQLN protein from H4 parental cells or cells FBXO42 K/O. Input samples before immunoprecipitation are indicated – bottom panel . C. Schematic representation of the E-Stub proximity ubiquitin labelling and enrichment. BirA-FBXO42 was co-expressed with a biotin acceptor ubiquitin mutant (bio GEF UBnc) followed by biotin treatment. Nascent ubiquitinated substrates are thus biotinylated and immunoprecipitated. D. Immunoblotting pf PP4 complex after expression of (doxycycline-inducible) BirA– FBXO42 and biotin bio GEF UBnc-Ub in T-REx™ Hek293 cells post. Proteins were induced by doxycycline for 24 hours and supplemented with biotin for 3 hours with or without MG132/MLN4924 as indicated, before protein lysis and immunoprecipitation. Input samples indicated in top panel.

Journal: bioRxiv

Article Title: Pervasive phenotypic effects of FBXO42 controlled by regulation of PP4 phosphatase

doi: 10.1101/2025.05.02.651838

Figure Lengend Snippet: A. Immunoblotting after expression of GFP-FBXO42, HA-ubiquitin and either Flag-PP4R2 or Flag-PP4C in HEK293T as indicated. Flag-PP4R2 or Flag-PP4C was isolated via Flag agarose beads pulldown before immunoblotting. Input samples are in the right panel. B. Immunoblotting after isolation of endogenous ubiquitinated proteins using recombinant GST-tagged UBA domain of UBIQLN protein from H4 parental cells or cells FBXO42 K/O. Input samples before immunoprecipitation are indicated – bottom panel . C. Schematic representation of the E-Stub proximity ubiquitin labelling and enrichment. BirA-FBXO42 was co-expressed with a biotin acceptor ubiquitin mutant (bio GEF UBnc) followed by biotin treatment. Nascent ubiquitinated substrates are thus biotinylated and immunoprecipitated. D. Immunoblotting pf PP4 complex after expression of (doxycycline-inducible) BirA– FBXO42 and biotin bio GEF UBnc-Ub in T-REx™ Hek293 cells post. Proteins were induced by doxycycline for 24 hours and supplemented with biotin for 3 hours with or without MG132/MLN4924 as indicated, before protein lysis and immunoprecipitation. Input samples indicated in top panel.

Article Snippet: Lentivirus expression construct TRIPZ-bio GEF UBnc puro (TRIPZ-bio GEF -UBnc-PURO was a gift from Rosa Barrio & James Sutherland Addgene plasmid # 208044) were packaged in HEK293T cells by transfect with psPAX2, pMD2.G and pTAT (pcDNA1-Tat was a gift from Akitsu Hotta Addgene plasmid # 138478).

Techniques: Western Blot, Expressing, Ubiquitin Proteomics, Isolation, Recombinant, Immunoprecipitation, Mutagenesis, Lysis