tcf lef binding sites  (Addgene inc)

Journal: PLoS ONE

Article Title: A Novel Aminothiazole KY-05009 with Potential to Inhibit Traf2- and Nck-Interacting Kinase (TNIK) Attenuates TGF-β1-Mediated Epithelial-to-Mesenchymal Transition in Human Lung Adenocarcinoma A549 Cells

doi: 10.1371/journal.pone.0110180

Figure Lengend Snippet: (A) Effects of KY-05009 on cell viability and TCF4 transcriptional activity. Serum-deprived A549 cells were treated with TGF-β1 or its combination with KY-05009 for 48 h, and then cell viability and TOPflash luciferase activity were measured. FOPflash-normalized TOPflash luciferase activity was represented to the relative TCF/LEF luciferase activity. The expression of TNIK and β-catenin in cytosolic and nuclear fractions (B), and the protein levels of TCF4-interacting proteins c were measured by Western blot analysis and immunoprecipitation assay, respectively. Actin, histone H3, and IgG were used as loading controls. The expression of cytosol and nucleus proteins was normalized by actin and histone H3, respectively. Reported results are representatives of triplicate experiments. ### p <0.001 (versus ‘the control’); ** p <0.01, *** p <0.001 (versus ‘the group treated with TGF-β1 only’).

Article Snippet: A549 cells were trasfected using TOPflash TCF reporter plasmid (wild type TCF binding site, Millipore), FOPflash plasmid (mutant TCF binding site, Millipore) and Lipofectamine 2000 (Invitrogen) in an antibiotics-free medium.

Techniques: Activity Assay, Luciferase, Expressing, Western Blot, Immunoprecipitation

Journal: The Journal of Clinical Investigation

Article Title: N-cadherin upregulation mediates adaptive radioresistance in glioblastoma

doi: 10.1172/JCI136098

Figure Lengend Snippet: (A) Western blot showing expression changes of several N-cad binding catenins following 6–12 cycles of irradiation (5 Gy) in mGS cells. (B) Fluorescence microscopy shows that β-catenin (green) selectively coaccumulates with N-cad (red) on the cell surface of mGSRR but not mGS cells. Nuclei were counterstained with Hoechst 33342 (blue). Scale bars: 25 μm. (C) Wnt/β-catenin regulated transcriptional activity in mGS and mGSRR cells measured through transient transfection with a luciferase reporter driven by a WT (TOP) or mutant (FOP) TCF binding site. ***P < 0.001, 2-tailed Student’s t test. (D) TOP/FOP ratio showing Wnt/β-catenin activity in parental N-cad–overexpressing and N-cad–KO mGS cells. **P < 0.01, ***P < 0.001, Tukey’s HSD test. (E) Microarray analysis showing that mRNA expression of multiple Wnt target genes is suppressed in mGSRR compared with mGS cells. Each group contains 2 independent replicates (n = 2). (F) qRT/PCR showing that NeuroD1, Ngn1, and Brn3a mRNAs are reduced in mGSRR cells. Two-tailed Student’s t test. (G) Western blot showing expression change of β-catenin (pan and non-phospho), c-Myc, and Tuj1 by N-cad–overexpressing and N-cad–KO mGS cells. All blots show representative images (n = 3 or more).

Article Snippet: Luciferase reporter gene transfections were performed with lipofectamine using a reporter gene construct driven by a TCF binding site (M50 Super 8x TOP Flash, Addgene, #12456) and a negative control with a mutant TCF binding site (M51 Super 8x FOP Flash, Addgene, #12457), which were provided by Randall Moon (University of Washington, Seattle, Washington, USA).

Techniques: Western Blot, Expressing, Binding Assay, Irradiation, Fluorescence, Microscopy, Activity Assay, Transfection, Luciferase, Mutagenesis, Microarray, Quantitative RT-PCR, Two Tailed Test