3× mutated tcf-binding site (fopflash  (Promega)

Journal:

Article Title: Oncogenic Mutants of RON and MET Receptor Tyrosine Kinases Cause Activation of the ?-Catenin Pathway

doi: 10.1128/MCB.21.17.5857-5868.2001

Figure Lengend Snippet: (A) RON M1254T receptor mutant causes constitutive transactivation of Tcf consensus sequence (TOPFLASH)-driven transcription in MDCK cells. MDCK cells expressing RON WT or M1254T mutant were infected with adenovirus encoding β-Gal or DN Tcf-4, and 24 h later these cells were transfected with luciferase reporter plasmids containing WT (TOPFLASH) or mutated (FOPFLASH) Tcf promoter. Tcf activity was determined by a luciferase assay as described in Materials and Methods. Data were normalized for total protein concentration. Data for a negative control FOPFLASH are not shown. Luciferase activity was calculated in fold increase, where luciferase activity in cells expressing RON WT and β-Gal was taken for 1. Bar graph data are means ± standard errors of three independent experiments. (B) The increased level of c-myc and D1 expression in cells with mutated RON is mediated by Tcf. MDCK cells expressing RON WT or M1254T mutant were infected with adenovirus encoding β-Gal or DN Tcf-4. After 48 h the amount of c-myc and cyclin D1 was determined in total lysates from cells by Western blotting (WB) with anti-c-myc and anti-cyclin D1 antibodies. The β-actin panel serves as a control showing an equal amount of protein in each sample. Positions of molecular-weight markers are indicated on the right.

Article Snippet: The reporter plasmids 3× WT Tcf-binding site (TOPFLASH) and 3× mutated Tcf-binding site (FOPFLASH) were from Promega.

Techniques: Mutagenesis, Sequencing, Expressing, Infection, Transfection, Luciferase, Activity Assay, Protein Concentration, Negative Control, Western Blot, Molecular Weight

Journal:

Article Title: Oncogenic Mutants of RON and MET Receptor Tyrosine Kinases Cause Activation of the ?-Catenin Pathway

doi: 10.1128/MCB.21.17.5857-5868.2001

Figure Lengend Snippet: (A) MET M1268T receptor mutant causes constitutive transactivation of Tcf consensus sequence (TOPFLASH)-driven transcription in NIH 3T3 cells. Activity of Tcf-4 in NIH 3T3 cells expressing MET WT or M1268T was determined by luciferase assay as described in the legend to Fig. ​Fig.6A.6A. Luciferase activity in cells expressing MET WT and β-Gal was set equal to 1. Bar graph data are means ± standard errors of three independent experiments. (B) The increased level of c-myc and D1 expression in cells with mutated MET is mediated by Tcf. NIH 3T3 cells expressing MET WT or M1268T were infected with an adenovirus encoding β-Gal or DN Tcf-4. After 48 h the amount of c-myc and cyclin D1 was determined in total lysates from cells by Western blotting with anti-c-myc and anti-cyclin D1 antibodies. MET tyrosine phosphorylation was determined by anti-PY antibodies in MET IPs. To estimate the amount of the MET in precipitates, the blot was probed with anti-MET antibodies (upper band, immature MET [170 kDa]; lower band, mature MET [140 kDa]). Tyrosine phosphorylation of β-catenin was detected by Western blotting (WB) with anti-PY antibodies. The amount of β-catenin in precipitates was determined with anti-β-catenin antibodies. The β-actin panel serves as a control showing an equal amount of protein in each sample. Positions of molecular-weight markers are indicated on the right.

Article Snippet: The reporter plasmids 3× WT Tcf-binding site (TOPFLASH) and 3× mutated Tcf-binding site (FOPFLASH) were from Promega.

Techniques: Mutagenesis, Sequencing, Activity Assay, Expressing, Luciferase, Infection, Western Blot, Molecular Weight