gata-binding site oligonucleotide  (Promega)

Journal: The Journal of Experimental Medicine

Article Title: The Transcription Factor T-bet Regulates Mucosal T Cell Activation in Experimental Colitis and Crohn's Disease

doi: 10.1084/jem.20011956

Figure Lengend Snippet: Increased nuclear expression of T-bet in LP T cells from patients with Crohn's disease. (A) EMSA using nuclear extracts of purified LP T cells from Crohn's disease and control patients and a radiolabeled T-bet DNA binding site. The location of the T-bet signal is indicated. (B) Auto- and cross-competition assay. Specificity of the EMSA signal obtained with the T-bet–binding site was shown by autocompetition with unlabeled T-bet binding site, whereas cross-competition with unlabeled SP-1, μE5, and AP-2 sites did not affect the signal. (C) Analysis of T-bet expression in nuclear extracts of LP T cells (Crohn's disease: n = 3; controls: n = 2) by Western blot analysis. A high expression of T-bet was observed in patients with Crohn's disease but not control patients. A second independent experiments with four patients per group showed similar results (data not shown). (D) Intracellular staining for STAT-1 and T-bet by FACS ® using LP cells from patients with Crohn's disease or control patients, as indicated. One representative experiment out of three is shown.

Article Snippet: Synthetic, double-stranded oligonucleotides containing binding sites for SP-1, AP-2, μE5, and GATA-3 for EMSA were obtained from Santa Cruz Biotechnology, Inc. and MWG Biotech, respectively.

Techniques: Expressing, Purification, Control, Binding Assay, Competitive Binding Assay, Western Blot, Staining

Journal: The Journal of Experimental Medicine

Article Title: The Transcription Factor T-bet Regulates Mucosal T Cell Activation in Experimental Colitis and Crohn's Disease

doi: 10.1084/jem.20011956

Figure Lengend Snippet: Changes in Th1 and Th2 cytokine production by LP cells in T-bet–deficient mice. (A) Histologic analysis of the large bowel of T-bet knockout mice (KO), heterozygous mice (HET), and wild-type littermates (WT). There was no evidence for colitis in T-bet–deficient animals. Similarly, no evidence of intestinal inflammation was observed in the small bowel (data not shown). (B) T cell–enriched LPMCs were isolated from the small and large bowel of wild-type (WT) and T-bet–deficient (KO) mice as well as from heterozygous (HET) mice and stimulated with antibodies to CD3 and CD28. Supernatants were taken after 48 h and analyzed for cytokine content by specific ELISA. Two representative experiments (experiment 1: red bars; experiment 2: purple bars) out of four are shown. (C) Western blot analysis and EMSA analysis for GATA-3 expression in nuclear extracts of LPMC from wild-type and T-bet–deficient mice. There was an increased expression of GATA-3 in LPMCs from T-bet knockout mice. The staining for β-actin and the EMSA for SP1 served as loading controls.

Article Snippet: Synthetic, double-stranded oligonucleotides containing binding sites for SP-1, AP-2, μE5, and GATA-3 for EMSA were obtained from Santa Cruz Biotechnology, Inc. and MWG Biotech, respectively.

Techniques: Knock-Out, Isolation, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Staining