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 Communication and Signaling : CCS

Article Title: Atg5-deficient mesenchymal stem cells protect against non-alcoholic fatty liver by accelerating hepatocyte growth factor secretion

doi: 10.1186/s12964-024-01950-x

Figure Lengend Snippet: The deficiency of Atg5 enhances the secretion of HGF by promoting RE production in MSCs. A ELISA detection of various growth factors in the MSC supernatant, including Angiogenin, VEGF, HGF, bFGF, and Ang-2. B ELISA detection of the secretion of different factors in the supernatants of MSC shGfp and MSC shAtg5 . C ELISA detection of HGF secretion at different culture times in MSC shGfp and MSC shAtg5 . D qPCR analysis of HGF transcription in MSC shGfp and MSC shAtg5 . E Western blot analysis of the expression of HGF, P62, and LC3 in MSC shGfp and MSC shAtg5 . F MSC shGfp and MSC shAtg5 treated with 3-MA and CQ, followed by Western blot detection of HGF expression. G-I Representative images of immunofluorescence staining analyzing the expression of Rab5a, Rab11a, LAMP1, and HGF. Scale bar = 25 μm. J Western blot analysis of the expression of endosome sorting-related proteins (EEA1, Rab11a, Rab5a, Rab7a, and LAMP1). For all panels, the data are presented as mean ± S.E.M, and statistical significance is indicated as * P < 0.05, ** P < 0.01, *** P < 0.001. ELISA, Enzyme linked immunosorbent assay; HGF, hepatocyte growth factor; RE, recycling endosome; 3-MA, 3-methyladenine; CQ, chloroquine

Article Snippet: Additionally, a primary antibody to HGF (26,881–1-AP), EEA1 (68,065–1-Ig), Rab11a (67,902–1-Ig), Rab5a (66,339–1-Ig), Rab7a (55,469–1-AP), and LAMP1 (67,300–1-Ig) were purchased from Proteintech.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Immunofluorescence, Staining

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