lentivirus expression construct tripz-bio gef ubnc puro  (Addgene inc)

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

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