Journal:

Article Title: Novel TFAP2B Mutations That Cause Char Syndrome Provide a Genotype-Phenotype Correlation

doi:

Figure Lengend Snippet: Clustal W alignment of human TFAP2 protein sequences. A, Alignment of the basic domains of the TFAP2 proteins. Numbering is according to the TFAP2B amino acid sequence. Identical amino acid residues are shaded in gray. The three novel and two previously reported mutations are indicated. B, Alignment of a portion of the transactivation domains that includes the PY motif. The P62R mutation is indicated.

Article Snippet: Electromobility Shift Assays (EMSAs) TFAP2 proteins were used in EMSAs with the MT2A −180 TFAP2 binding sequence (5′-GAACTGACCGCCCGCGGCCCGTGTGCAGAG-3′) (Promega) that had been end-labeled with 32 P, using the T4 polynucleotide kinase.

Techniques: Sequencing, Mutagenesis

Journal:

Article Title: Novel TFAP2B Mutations That Cause Char Syndrome Provide a Genotype-Phenotype Correlation

doi:

Figure Lengend Snippet: Expression and function of recombinant TFAP2B proteins. Left, Autoradiogram of an SDS-PAGE with expressed wild-type (wt) and mutant TFAP2B proteins. Mobility of proteins of varying mass are indicated at the right. Right, Autoradiogram of an EMSA performed using the recombinant TFAP2B proteins that had been incubated with [32P]-labeled DNA with the consensus TFAP2 binding sequence. Free probe is indicated at bottom.

Article Snippet: Electromobility Shift Assays (EMSAs) TFAP2 proteins were used in EMSAs with the MT2A −180 TFAP2 binding sequence (5′-GAACTGACCGCCCGCGGCCCGTGTGCAGAG-3′) (Promega) that had been end-labeled with 32 P, using the T4 polynucleotide kinase.

Techniques: Expressing, Recombinant, SDS Page, Mutagenesis, Incubation, Labeling, Binding Assay, Sequencing

Journal:

Article Title: Novel TFAP2B Mutations That Cause Char Syndrome Provide a Genotype-Phenotype Correlation

doi:

Figure Lengend Snippet: EMSA with cotranslated TFAP2B and truncated TFAP2A proteins. Truncated TFAP2A (ΔN165), which retains dimerization and DNA-binding properties, was cotranslated with wild-type and mutant TFAP2B. TFAP2 proteins were incubated with [32P]-labeled DNA with the consensus TFAP2 binding sequence and electrophoresed. The two homodimer species (upper and lower shifted complexes) and the heterodimer (intermediate shifted complex) are indicated.

Article Snippet: Electromobility Shift Assays (EMSAs) TFAP2 proteins were used in EMSAs with the MT2A −180 TFAP2 binding sequence (5′-GAACTGACCGCCCGCGGCCCGTGTGCAGAG-3′) (Promega) that had been end-labeled with 32 P, using the T4 polynucleotide kinase.

Techniques: Binding Assay, Mutagenesis, Incubation, Labeling, Sequencing

Journal:

Article Title: Novel TFAP2B Mutations That Cause Char Syndrome Provide a Genotype-Phenotype Correlation

doi:

Figure Lengend Snippet: Transient expression of wild-type or mutant TFAP2B in NIH3T3 cells. Cells were transfected with 1.5 mg of the CAT reporter construct (A2BCAT) with three copies of TFAP2 binding sequence or an equivalent amount of the CAT-only construct (BCAT). To test for transactivation, 0.3 mg wild-type (wt) or mutant TFAP2B construct was cotransfected with A2BCAT. After 48 h, cells were lysed, and the CAT concentrations in the lysates were determined. To normalize for transfection efficiency, 0.5 mg of pQB125 was cotransfected, and GFP fluorescence was measured. The bars indicate the mean and standard errors from three independent transfections. Units are arbitrary, and the mean from the condition with only A2BCAT was set at 1.0.

Article Snippet: Electromobility Shift Assays (EMSAs) TFAP2 proteins were used in EMSAs with the MT2A −180 TFAP2 binding sequence (5′-GAACTGACCGCCCGCGGCCCGTGTGCAGAG-3′) (Promega) that had been end-labeled with 32 P, using the T4 polynucleotide kinase.

Techniques: Expressing, Mutagenesis, Transfection, Construct, Binding Assay, Sequencing, Fluorescence