mouse stromal fibroblast mef bl  (ATCC)

Journal: Frontiers in Immunology

Article Title: Mouse TAPBPR shows functional similarity to human TAPBPR in shaping the MHC-I immunopeptidome

doi: 10.3389/fimmu.2026.1756668

Figure Lengend Snippet: Feature of mouse TAPBPR protein. (A) ClustalWS alignment comparison between mouse TAPBPR (mouse TAPBPR, UniProt Q8VD31 ) and human TAPBPR (UniProt Q9BX59 ) proteins. Blosum62 scoring system was generated by Jalview 2.11.4.0 software. Boxes highlight the peptide editing loop (blue), MHC-I binding sites characterized as TN5, TN6, TC2 and TC3 (red), the free cysteine residue (black), two predicted N-linked glycosylation sites in mouse TAPBPR (purple, with the asparagine indicated by an asterisk) and the cytoplasmic tail regions (yellow). The endogenous mouse TAPBPR sequence in MC-38, B16-F10, and MEF-BL/6–1 cells was confirmed as equivalent to the UniProt reference. (B) Predicted AlphaFold2 structure of mouse TAPBPR (green) bound to H2-D b (blue) with N-linked glycosylations (pink) modelled using GLYCAM ( https://glycam.org ). For H2-D b , only two of the three glycans are visible in the image, with N86 obscured by the orientation depicted. (C) Representative histograms and bar graphs showing mean fluorescence intensity (MFI) of intracellular TAPBPR expression, detected using AnDi3 antibody, on IFNγ-treated wildtype (WT) MC-38, B16-F10, and MEF-BL/6–1 cells compared to TAPBPR knockout (KO) and mouse TAPBPR overexpressed (OE) equivalents, which serve as negative and positive controls, respectively. Error bars show MFI -/+ standard error of mean (SEM) from three independent experiments. *p ≤ 0.05, **p ≤ 0.01 using unpaired t-test. (D) Histograms showing IFNγ inducibility of intracellular TAPBPR expression in WT MC-38 and B16-F10 cells and in cells transduced to overexpress (OE) mouse TAPBPR.

Article Snippet: Murine colon adenocarcinoma MC-38 (Kerfast, Newark, CA 94560, USA), mouse melanoma B16-F10, Lewis lung carcinoma LL/2 and mouse stromal fibroblast MEF-BL/6-1 (ATCC SCRC-1008) cells were maintained in Dulbecco’s Modified Eagle’s medium (DMEM)(CAT: 41966052, GibcoTM, Thermo Fisher Scientific, Paisley, Renfrewshire, UK), supplemented with 10% fetal bovine serum (FBS)(CAT: 10500064, GibcoTM) and 100 units/mL penicillin-streptomycin (CAT: 15140122, GibcoTM) at 37 °C, 5% CO 2 , and humid atmosphere.

Techniques: Comparison, Generated, Software, Binding Assay, Residue, Glycoproteomics, Sequencing, Fluorescence, Expressing, Knock-Out

Journal: Frontiers in Immunology

Article Title: Mouse TAPBPR shows functional similarity to human TAPBPR in shaping the MHC-I immunopeptidome

doi: 10.3389/fimmu.2026.1756668

Figure Lengend Snippet: Mouse TAPBPR interaction partners identified in B16-F10, MC-38 and MEF-BL/6–1 cell lines. Mouse TAPBPR was isolated by immunoprecipitation, using Andi38 antibody, from TAPBPR knockout (KO) or mouse TAPBPR overexpressing (OE) from (A) B16-F10 cells, (B) MC-38 cells, (C) MEF-BL/6–1 cells or (D) MC-38 cells with β2m knocked out. Scatterplots show all proteins identified via mass spectrometry in the mouse TAPBPR pull-downs in cells overexpressing mouse TAPBPR compared to the equivalent TAPBPR KO cell line. Selected significant interaction partners highlighted are TAPBPR (pink), H2-D b (red), H2-K b (yellow), MHC-I (orange), which covers peptides common to H2 molecules and therefore cannot be assigned to a specific MHC-I molecule, β2m (navy) and known components of the MHC-I antigen presentation pathway (purple). (E) Confirmation of mouse TAPBPR binding partners at endogenous TAPBPR levels in MC-38 cells. Immunoblots indicating abundance of mouse TAPBPR (mTAPBPR), MHC-I, β2m, calnexin, tapasin, TAP2, and GAPDH (loading control) in the whole cell lysates and mouse TAPBPR immunoprecipitates (IP: mTAPBPR) from WT MC-38 cells. MC-38 with TAPBPR knocked out (KO) or overexpressing mouse TAPBPR (OE) are included as controls. Cells competent for β2m expression or with β2m knocked down (β2m KD) were compared to assess the importance of the TAPBPR/MHC-I interaction in the observed associations. An antibody-only lane is included to highlight the antibody’s heavy chain used in the immunoprecipitation. N = 1, for tapasin and TAP2 blot. (F) Endogenously expressed mouse TAPBPR exhibits a prolonged association with H2-D b compared to H2-K b in both MC-38 and B16 cells. Immunoblots indicating abundance of mTAPBPR, MHC-I, β2m, calnexin, and GAPDH (loading control) in the whole cell lysate and mTAPBPR immunoprecipitated fraction (IP: mTAPBPR) with Andi 38 from MC-38 or B16-F10 WT cells, and variant cell lines expressing H2-D b only (H2-K b knockout), H-2K b only (H2-D b knockout) or lacking efficient expression of both H2-D d and -K b following β2m knock down (KD). Representative of three independent experiments. Note: Arrowheads indicate the positioning of the major TAPBPR and MHC-I bands in the gels, where background bands were present in the immunoprecipitations. The position of TAPBPR relative to the antibody control also varies due to minor changes in running conditions between experiments. Note: WT cells in F were treated with a non-targeting RNA guide in the RNP.

Article Snippet: Murine colon adenocarcinoma MC-38 (Kerfast, Newark, CA 94560, USA), mouse melanoma B16-F10, Lewis lung carcinoma LL/2 and mouse stromal fibroblast MEF-BL/6-1 (ATCC SCRC-1008) cells were maintained in Dulbecco’s Modified Eagle’s medium (DMEM)(CAT: 41966052, GibcoTM, Thermo Fisher Scientific, Paisley, Renfrewshire, UK), supplemented with 10% fetal bovine serum (FBS)(CAT: 10500064, GibcoTM) and 100 units/mL penicillin-streptomycin (CAT: 15140122, GibcoTM) at 37 °C, 5% CO 2 , and humid atmosphere.

Techniques: Isolation, Immunoprecipitation, Knock-Out, Mass Spectrometry, Immunopeptidomics, Binding Assay, Western Blot, Control, Expressing, Variant Assay, Knockdown