Journal: Nature immunology
Article Title: Priming for T helper type 2 differentiation by interleukin 2-mediated induction of IL-4 receptor ? chain expression
Figure Lengend Snippet: STAT5-dependent regulation of IL-4Rα expression. ( a ) IL-4Rα expression on splenic CD4 + T cells from wild-type and Stat5b transgenic mice 40 , as evaluated by flow cytometry. The experiment shown is representative of two independent experiments with 2 to 3 mice in each group. ( b ) Expression of indicated transcripts, after Cre recombinase-mediated deletion of the LoxP-flanked Stat5a / Stat5b locus 29 in splenic T cells that then were cultured with IL-2. See Supplementary Table 1 , online for the entire list of genes. Three independent experiments were performed. ( c ) Schematic of five TTCN 3 GAA potential GAS motifs in the mouse Il4ra gene 5′ regulatory region and first intron. GAS1 is approximately 3.5 kb (mouse) or 1.5 kb (human) 5′ of the Il4ra transcription initiation site (TIS), whereas GAS2, GAS3, GAS4, and GAS5 are in the first intron. ( d ) Indicated PCR-generated constructs (left; luc , luciferase) were transfected into YT cells not treated or treated with 100 U/ml of IL-2 and cell lysates were analyzed for luciferase activity (right). Three independent experiments were performed. ( e ) EMSA 41 using an Il4ra probe spanning GAS3 and nuclear extracts 41 from human peripheral T cells. Cells were untreated or treated with IL-2 or IL-6. For supershifting assays, each antiserum was pre-incubated with nuclear extracts before adding labeled probe. In lane 6, a probe mutated at GAS3 was the control. The experiment shown is representative of three independent experiments. ( f ) ChIP assays 41 of STAT5A and STAT5B binding using CD4 + splenic T cells from Balb/c mice pre-activated with anti-CD3 and anti-CD28 for 3 days, rested overnight, not treated or treated with 100 U/ml IL-2 for 4-5 h at 37 °C, followed by cross-linking with formaldehyde. Nuclear lysates were immunoprecipitated at 4°C overnight with anti-STAT5A, anti-STAT5B (R D Systems) or an isotype control antibody to allow normalization of the fold induction by IL-2. After deproteination and cross-link reversal, selected DNA sequences were assessed by real-time PCR. Primers spanning the Socs3 STAT binding site were used as a positive control and Gapdh as a negative control. See Supplementary Table 10 for sequences or primers used in ChIP experiments. The experiment shown is representative of three independent experiments.
Article Snippet: Animal protocols were approved by the NHLBI Animal Care and Use Committee and followed the NIH Guidelines “Using Animals in Intramural Research.” Splenic total T cells or CD4+ subpopulations were purified from 5-12 week old mice by negative or positive selection using magnetic beads (Miltenyi), cultured in RPMI 1640 medium containing 10 mM Hepes (pH 7.0), 10% fetal bovine serum, 2 mM L-glutamine, and antibiotics (complete medium), and activated for 1.5 h at 37°C in dishes pre-coated with anti-CD3 (2 μg/ml in PBS) in complete medium containing 1 μg/ml anti-CD28 (PharMingen).
Techniques: Expressing, Transgenic Assay, Mouse Assay, Flow Cytometry, Cytometry, Cell Culture, Polymerase Chain Reaction, Generated, Construct, Luciferase, Transfection, Activity Assay, Incubation, Labeling, Chromatin Immunoprecipitation, Binding Assay, Immunoprecipitation, Real-time Polymerase Chain Reaction, Positive Control, Negative Control