Journal:

Article Title: Measles Virus (MV) Nucleoprotein Binds to a Novel Cell Surface Receptor Distinct from Fc?RII via Its C-Terminal Domain: Role in MV-Induced Immunosuppression

doi: 10.1128/JVI.77.21.11332-11346.2003

Figure Lengend Snippet: MV-N binding on different cell types

Article Snippet: The cell lines 3D1 (murine cortical TEC), HeLa (epithelial human carcinoma from cervix), MeWo (fibroblastic human melanoma from lymph node), NIH 3T3 (mouse fibroblasts from embryo), Jurkat (human acute T-cell leukemia), and Vero (monkey fibroblast from kidney) were obtained from the American Type Culture Collection (Manassas, Va.).

Techniques: Binding Assay, Inhibition, Expressing

Journal: Clinical and Developmental Immunology

Article Title: Modulatory Effect of 1,25-Dihydroxyvitamin D 3 on IL1 β -Induced RANKL, OPG, TNF α , and IL-6 Expression in Human Rheumatoid Synoviocyte MH7A

doi: 10.1155/2013/160123

Figure Lengend Snippet: Effect of 1,25(OH) 2 D 3 on IL β -induced TNF α and IL-6 production in MH7A. MH7A cells were stimulated with 20 ng/mL IL1 β and then treated with different concentrations of 1,25(OH) 2 D 3 (0.1 nM, 1 nM, 10 nM, and 100 nM) for 48 h. The effects of 1,25(OH) 2 D 3 on IL β -induced TNF α (a) and IL-6 (b) mRNA expression in MH7A was analyzed by real-time PCR, and the production of IL-6 in supernatants was shown in (c). The data shown are the mean ± SD for three independent experiments, each in triplicate. * P < 0.05 compared to cells cultured with IL-1 β alone. ** P < 0.01 compared to cells cultured with IL-1 β alone.

Article Snippet: Cytokine production was determined in cell culture supernatants using ELISA specific for human IL-6 and TNF α (BOSTER-BIO; Wuhan; China) following manufacturer's guidelines.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Cell Culture

Journal: Nature immunology

Article Title: Recruitment of calcineurin to the T cell receptor positively regulates T cell activation

doi: 10.1038/ni.3640

Figure Lengend Snippet: TCR-induced pathways are differentially phosphorylated by CsA and FK506. (a–j) Immunoblot analysis of lysates from Jurkat cells (a,c,e,g,i) and human CD4+ T cells (b,d,f,h,j) that were pretreated with medium, FK506, or CsA at 37 °C and then activated with soluble OKT3 (anti-CD3) and anti-mouse-IgG for the indicated times. After probing for phosphoproteins (designated by the prefix ‘p-’), membranes were stripped and reblotted to visualize the total amounts of the cellular proteins corresponding to the phosphoproteins that were probed. (k) Immunoblot analysis of whole-cell lysates from ZAP70-deficient P116 cells that were retrovirally transduced with ZAP70WT- or ZAP70Y493F-expressing constructs and stimulated for the indicated times. The data in a–k are representative of three independent experiments with similar results.

Article Snippet: Human CD4 + T cells were isolated from buffy coats of healthy volunteers (NIH Blood Bank) using the human CD4 cell recovery column kit (Cedarlane), according to the manufacturer’s instructions.

Techniques: Western Blot, Transduction, Expressing, Construct

Journal: Nature immunology

Article Title: Recruitment of calcineurin to the T cell receptor positively regulates T cell activation

doi: 10.1038/ni.3640

Figure Lengend Snippet: Knockdown of calcineurin expression in T cells recapitulates the effects of the drugs. (a,b) Top, representative immunoblot analysis for the expression of calcineurin in lysates from Jurkat T cells (a) or human CD4+ T cells (b) that were untransfected or electroporated with a control (Ctrl), calcineurin A α-specific, or calcineurin A β-specific siRNA either individually or in combination 72 h after transfection. Bottom, quantification of calcineurin expression relative to ZAP70 levels. In b, the lanes were rearranged for clarity. (c,d) Representative western blot analysis of lysates from siRNA-treated Jurkat cells (c) and human CD4+ T cells (d) after stimulation with OKT3 and anti-mouse-IgG for the indicated times. Stripped membranes were reblotted to ascertain total amounts of the indicated cellular proteins. Data are representative of three independent experiments. (e) Immunoblot analysis of Jurkat cells expressing endogenous or endogenous plus codon-optimized calcineurin A (calcineurin Aopt) after activation with OKT3 for 5 min. The data are representative of three (c,d) or two (e) independent experiments with similar results.

Article Snippet: Human CD4 + T cells were isolated from buffy coats of healthy volunteers (NIH Blood Bank) using the human CD4 cell recovery column kit (Cedarlane), according to the manufacturer’s instructions.

Techniques: Expressing, Western Blot, Transfection, Activation Assay

Journal: Nature immunology

Article Title: Recruitment of calcineurin to the T cell receptor positively regulates T cell activation

doi: 10.1038/ni.3640

Figure Lengend Snippet: Association of calcineurin with signaling components requires LCK activity and intact ZAP70. (a,b) Immunoblot analysis for co-immunoprecipiation of calcineurin after immunoprecipitation of TCR-ζ from activated Jurkat cells (a) and human CD4+ T cells (b) that were treated as indicated (with or without FK506 or CsA) and stimulated with anti-CD3 for the indicated times. (c) Immunoblot analysis of TCR-ζ immunoprecipitates from Jurkat cells that were incubated with or without the Src kinase family inhibitor PP1 and activated for different amounts of times. Whole-cell lysates (WCL) from these cells were subjected to immunoblot analysis with a phospho-LCKY394-specific antibody to confirm inhibition of kinase activity. (d,e) Immunoblot analysis of anti-calcineurin immunoprecipitates from Jurkat cells (d) and human CD4+ T cells (e) that were activated for the indicated times. Stripped membranes were reprobed with anti-calcineurin to ensure equal amounts of loading of the precipitated complex. (f,g) Immunoblotting of anti-TCR-ζ immunoprecipitates from Jurkat cells and ZAP70-deficient P116 cells (f) or from Jurkat cells that were or were not treated with the ZAP70 kinase inhibitor piceatannol (g) following activation for different amounts of times. Whole-cell lysates were subjected to immunoblot analysis with anti-phospho-LATY171 to confirm inhibition of ZAP70 activity. (h,i) Immunoblot analysis of anti-calcineurin-A (or an isotype control) (h) or anti-TCR-ζ (i) immunoprecipitates from Jurkat cells that were unactivated or activated for the indicated amounts of times. The data in a–i are representative of three independent experiments with similar results.

Article Snippet: Human CD4 + T cells were isolated from buffy coats of healthy volunteers (NIH Blood Bank) using the human CD4 cell recovery column kit (Cedarlane), according to the manufacturer’s instructions.

Techniques: Activity Assay, Western Blot, Immunoprecipitation, Incubation, Inhibition, Activation Assay

Journal: Nature immunology

Article Title: Recruitment of calcineurin to the T cell receptor positively regulates T cell activation

doi: 10.1038/ni.3640

Figure Lengend Snippet: phospho-LCKS59 is a substrate of calcineurin in vivo and in vitro. (a,b) Immunoblot analysis of Jurkat cells (a) and primary human CD4+ T cells (b) that were treated with either medium or the indicated compounds and stimulated with anti-CD3 for the indicated amounts of time. (c) Immunoblot analysis of anti-TCR-ζ immunoprecipitates from Jurkat cells after activation for the indicated amounts of time. (d,e) Immunoblot analysis of proteins from Jurkat cells (d) and human CD4+ T cells (e) that were transfected with a control siRNA or with PPP3CA-specific and PPP3CB-specific siRNAs and that were activated with anti-CD3 for the indicated amounts of time. (f) Immunoblot analysis of TCR-ζ immune-complexes from Jurkat cells that were activated in the absence or presence of FK506 for the indicated times and subjected to an in vitro phosphatase assay using recombinant human calcineurin (rhCN). The data in a–f are representative of three independent experiments.

Article Snippet: Human CD4 + T cells were isolated from buffy coats of healthy volunteers (NIH Blood Bank) using the human CD4 cell recovery column kit (Cedarlane), according to the manufacturer’s instructions.

Techniques: In Vivo, In Vitro, Western Blot, Activation Assay, Transfection, Phosphatase Assay, Recombinant

Journal: Nature immunology

Article Title: Recruitment of calcineurin to the T cell receptor positively regulates T cell activation

doi: 10.1038/ni.3640

Figure Lengend Snippet: Calcineurin’s effect on inhibitory phospho-LCKS59 promotes TCR-induced LFA-1-mediated cell adhesion. (a,b) Quantification of Jurkat (a) and human CD4+ T (b) cells, as assessed by trypan blue exclusion, that bound to ICAM1-coated plates after treatment with the indicated compounds for 30 min at 37 °C and stimulation with anti-CD3 for an additional 30 min. The percentage binding was calculated for each relative to the activated sample, which was considered to be 100%. (c) Immunoblot analysis of human CD4+ T cells that were treated as indicated and activated with anti-CD3. (d) Numbers OR Quantification of T cells from AND TCR-transgenic mice that bound to ICAM1-coated plates after incubation with MCC-pulsed APCs (I-Ek- and ICAM1-expressing DCEK cells) in the presence of the indicated reagents. (e) Quantification of ICAM1-bound JCam1.6 cells stably expressing LCK, LCKS59A, or LCKS59E that bound to ICAM1-coated plates after activation with anti-CD3 in the presence of FK506 or CsA for 30 min at 37 °C. The data in a,b,e are mean ± s.e.m. and are representative of three (a,b) or four (e) independent experiments. In d, the data are mean ± s.e.m. of four independent experiments. *P < 0.05; **P < 0.01; ***P < 0.005; ****P < 0.0001; n.s., not significant; by paired t-test.

Article Snippet: Human CD4 + T cells were isolated from buffy coats of healthy volunteers (NIH Blood Bank) using the human CD4 cell recovery column kit (Cedarlane), according to the manufacturer’s instructions.

Techniques: Binding Assay, Western Blot, Transgenic Assay, Incubation, Expressing, Stable Transfection, Activation Assay