Journal: The Journal of Biological Chemistry

Article Title: A Soluble Fragment of the Tumor Antigen BCL2-associated Athanogene 6 (BAG-6) Is Essential and Sufficient for Inhibition of NKp30 Receptor-dependent Cytotoxicity of Natural Killer Cells *

doi: 10.1074/jbc.M113.483602

Figure Lengend Snippet: BAG-6686–936 inhibits NK cell activation and cytotoxicity. A, scheme of the experimental setup using NKp30-transduced A5-GFP reporter cells for detection of NKp30-dependent CD3ζ-signaling based on induced GFP expression. B, NKp30- or mock-transduced A5-GFP reporter cells were cultivated in wells with immobilized proteins and analyzed for the number of GFP positive cells. C, NKp30-transduced A5-GFP reporter cells were co-cultivated with Ba/F3 cells transduced with the NKp30 ligand B7-H6 (Ba/F3-B7-H6). GFP expression was analyzed in the absence or presence of recombinant BAG-6686–936, NKp30-IgG1-Fc, IFNAR2-IgG1-Fc, or an anti-NKp30 antibody. D, CD107a surface expression, a marker for NK cell cytotoxicity, was measured by flow cytometry after co-incubation of NK-92 cells with Ba/F3-B7-H6 cells or mock-transduced Ba/F3 cells (Ba/F3-GFP). For reference, cells were treated with phorbol 12-myristate 13-acetate (PMA) and ionomycin (iono.). E, BAG-6686–936 and control proteins were probed for inhibition of Ba/F3-B7-H6 cell-induced CD107a surface expression by flow cytometry. F, in parallel, NK-92 cells were permeabilized and analyzed for intracellular IFN-γ expression after co-incubation with Ba/F3-B7-H6 cells and Ba/F3-GFP cells or treatment with phorbol 12-myristate 13-acetate and ionomycin. G, IFN-γ expression of NK-92 cells, stimulated with Ba/F3-B7-H6 cells, was analyzed by flow cytometry in the presence of recombinant proteins. Data points represent mean values ± S.E. obtained from least five individual experiments performed; *, p < 0.01; **, p < 0.001; ***, p < 0.0001 by Student's t test. ns, not significant.

Article Snippet: NK and target cells (Ba/F3-B7-H6, Ba/F3-GFP) were mixed at an effector:target ratio of 1:1, and cells were incubated for 1 h at 37 °C in the presence of anti-human CD107a pacific blue-conjugated antibodies (Miltenyi Biotech).

Techniques: Activation Assay, Expressing, Transduction, Recombinant, Marker, Flow Cytometry, Incubation, Control, Inhibition

Journal: Cell

Article Title: A membrane-associated MHC-I inhibitory axis for cancer immune evasion

doi: 10.1016/j.cell.2023.07.016

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: CD107a (LAMP-1) Antibody, anti-human, PE, REAfinity TM , Miltenyi Biotec , Cat# 130–111-621; RRID: AB_2654474.

Techniques: Control, Purification, Blocking Assay, Virus, Recombinant, Protease Inhibitor, Transfection, Immunoprecipitation, Activation Assay, Magnetic Beads, Reverse Transcription, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, Double Knockout, shRNA, Real-time Polymerase Chain Reaction, Genome Wide, Plasmid Preparation, Software, Flow Cytometry