hgitrl  (Miltenyi Biotec)

Journal: Cellular and Molecular Immunology

Article Title: An isoleucine-zipper motif enhances costimulation of human soluble trimeric GITR ligand

doi: 10.1038/cmi.2010.7

Figure Lengend Snippet: Figure 3 ILZ-hsGITRL proteins possess stronger binding activities and costimulation. (a, b) Binding activities of hsGITRL and ILZ-hsGITRL with anti-hGITRL, were performed by ELISA (a) and FCM (b). CD4+ T cells were stimulated with antihuman CD3 mAb for 24 h, and cultured with control proteins (gray histograms), hsGITRL (dotted lines) and ILZ-hsGITRL (solid lines) proteins, and stained with FITC-conjugated anti-hGITRL mAb. Data shown were the means (±SD) of triplicate cultures or representatives of three independent assays. (c) Comparing with hsGITRL, the trimer of ILZ-hsGITRL enhanced CD4+ T cells' proliferative response to different protein concentrations, respectively. Data shown were the means (±SD) of triplicate cultures and representatives of three independent assays. (d) Levels of IFN-γ in the culture supernatants were measured by ELISA kit. Data shown were the means (±SD) of triplicate cultures. (e) CD4+ T cells were stimulated with ILZ-hsGITRL or hsGITRL (5.0 µg/ml) in the presence of antihuman CD3 mAb. Cellular lysates were collected at indicated time points and the levels of ERK1/2 phosphorylation were analyzed by western blotting. The experiments were repeated three times with essentially the same results. ERK1/2, extracellular signal-regulated kinase-1/2; FCM, flow cytometry; hsGITRL, human soluble glucocorticoid-induced tumor-necrosis factor receptor ligand; IFN-γ, interferon-γ ILZ-hsGITRL, isoleucine-zipper hsGITRL; mAb, monoclonal antibody.

Article Snippet: The soluble proteins were produced, obtained by ultrasound sonication on the ice, and then identified by anti-hGITRL mAb (eBioscience, San Diego, CA, USA) and anti-His mAb (Cell Signal Technology, Danvers, MA, USA).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Cell Culture, Control, Staining, Phospho-proteomics, Western Blot, Flow Cytometry

Journal: Cellular and Molecular Immunology

Article Title: An isoleucine-zipper motif enhances costimulation of human soluble trimeric GITR ligand

doi: 10.1038/cmi.2010.7

Figure Lengend Snippet: Figure 3 ILZ-hsGITRL proteins possess stronger binding activities and costimulation. (a, b) Binding activities of hsGITRL and ILZ-hsGITRL with anti-hGITRL, were performed by ELISA (a) and FCM (b). CD4+ T cells were stimulated with antihuman CD3 mAb for 24 h, and cultured with control proteins (gray histograms), hsGITRL (dotted lines) and ILZ-hsGITRL (solid lines) proteins, and stained with FITC-conjugated anti-hGITRL mAb. Data shown were the means (±SD) of triplicate cultures or representatives of three independent assays. (c) Comparing with hsGITRL, the trimer of ILZ-hsGITRL enhanced CD4+ T cells' proliferative response to different protein concentrations, respectively. Data shown were the means (±SD) of triplicate cultures and representatives of three independent assays. (d) Levels of IFN-γ in the culture supernatants were measured by ELISA kit. Data shown were the means (±SD) of triplicate cultures. (e) CD4+ T cells were stimulated with ILZ-hsGITRL or hsGITRL (5.0 µg/ml) in the presence of antihuman CD3 mAb. Cellular lysates were collected at indicated time points and the levels of ERK1/2 phosphorylation were analyzed by western blotting. The experiments were repeated three times with essentially the same results. ERK1/2, extracellular signal-regulated kinase-1/2; FCM, flow cytometry; hsGITRL, human soluble glucocorticoid-induced tumor-necrosis factor receptor ligand; IFN-γ, interferon-γ ILZ-hsGITRL, isoleucine-zipper hsGITRL; mAb, monoclonal antibody.

Article Snippet: The purified anti-hGITRL mAbs were coated onto a 96-well plate (Corning, New York, USA) overnight at 4 °C.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Cell Culture, Staining, Western Blot, Flow Cytometry