Journal: Scientific reports

Article Title: A 3 base pair deletion in TBX1 leads to reduced protein expression and transcriptional activity.

doi: 10.1038/srep44165

Figure Lengend Snippet: Figure 1. Characteristics of the mutation c.303-305delGAA in TBX1. (A) Chromatograms of the homozygous mutation found in the TOF patient of cohort 1; (B) Structural representations of the mutation in TBX1 gene and protein; (C) Homology analysis of the 102 K position of TBX1 protein across different species; (D) the prediction result of MutationTaster. (*The probability value is the probability of the prediction, and a value close to 1 indicates a high ‘security’ of the prediction). (E) Chromatograms of the heterozygous c.303- 305delGAA mutation found in cohort 2.

Article Snippet: The human TBX1 isoform C cDNA clone was purchased from OriGene.

Techniques: Mutagenesis

Journal: Scientific reports

Article Title: A 3 base pair deletion in TBX1 leads to reduced protein expression and transcriptional activity.

doi: 10.1038/srep44165

Figure Lengend Snippet: Figure 2. Subcellular localization of wild-type and mutant TBX1 in transiently transfected COS7 cells. Cells were co-stained with 4,6-diamino-2-phenylindole (DAPI) to illustrate nuclei. Wild-type TBX1 and the p.102delK mutant both localize to the nucleus.

Article Snippet: The human TBX1 isoform C cDNA clone was purchased from OriGene.

Techniques: Mutagenesis, Transfection, Staining

Journal: Scientific reports

Article Title: A 3 base pair deletion in TBX1 leads to reduced protein expression and transcriptional activity.

doi: 10.1038/srep44165

Figure Lengend Snippet: Figure 3. The mutant protein TBX1102delK showed decreased expression level, and lost transcriptional activity. (A,B) Real-time qPCRs show the mRNA level of the mutant TBX1c.303_305delGAA was similar with that of the wild-type TBX1 in both 3T3 and C2C12 cells. (C,D) Western blots show the protein level of the mutant TBX1102delK and wild-type TBX1. The mutant showed reduced protein level compared with the wild-type in both 3T3 and C2C12 cells. The full-length blots are presented in the Supplementary Figure S1 and S2, respectively. (E,F) show that the 3T3 or C2C12 cells were co-transfected with the 4XT/2-minP reporter and either a pcDNA3.1(+) control vector (Blank), the TBX1 wild-type construct (WT), or the mutant TBX1c.303_305delGAA construct (Mut1). The results were normalized for transfection efficiency to a co-transfected pGL4.74[hRluc/ TK] vector. The TBX1102delK mutant showed significantly reduced transcription activity compared withthe wild- type protein in both 3T3 (reduced 42.4% vs. TBX1wt, p = 0.0112) and C2C12 cells (reduced 19.9% vs. TBX1wt, p = 0.0164). (G) Shows the C2C12 cells were co-transfected with the FGF10luc reporter and either the blank, the wild-type, or the mutant constructs (Mut1 = TBX1102delK and Mut2 = TBX1E129K). The results were normalized for transfection efficiency to a co-transfected pGL-TK vector. The TBX1102delK and TBX1E129K mutants all showed significantly reduced transcription activity compared with the wild-type protein (reduced 22.9% vs. TBX1wt, p = 0.0050 and reduced 28.7% vs. TBX1wt, p = 0.0112, respectively). (H) shows that significantly reduced transcriptional activity of the TBX1102delK and TBX1E129K mutant proteins on the WNTluc1 reporter compared with the wild-type protein in C2C12 cells (reduced 35.5% vs. TBX1wt, p = 0.0037 and reduced 25.4% vs. TBX1wt, p = 0.0156, respectively). (I) shows that the transcription activity of the two mutant proteins also decreased significantly on the WNTluc2 reporter in C2C12 cells (reduced 34% vs. TBX1wt, p = 0.0259 and reduced 31% vs. TBX1wt, p = 0.0242, respectively). The results are all shown as the mean ± SEM of three independent experiments performed in triplicate. *p < 0.05; **p < 0.01; blank: pcDNA3.1(+) vector; WT: wild- type TBX1; mut1: the TBX1102delK mutant; mut2: the TBX1E129K mutant.

Article Snippet: The human TBX1 isoform C cDNA clone was purchased from OriGene.

Techniques: Mutagenesis, Expressing, Activity Assay, Western Blot, Transfection, Control, Plasmid Preparation, Construct

Journal: Scientific reports

Article Title: A 3 base pair deletion in TBX1 leads to reduced protein expression and transcriptional activity.

doi: 10.1038/srep44165

Figure Lengend Snippet: Figure 4. Electrophoretic mobility shift assay to study DNA-binding activity of wild-type and mutant TBX1. DNA binding assay show that both the wild-type and p.102delK mutant TBX1 could bind to DNA (lane 2 and 3). Specificity of the binding is confirmed by nuclear protein of HEK293T cell transfected with the pcDNA3.1(+) control vector (lane 1) and the addition of unlabled probe (100X) (lane 4). The full-length image is presented in the Supplementary Figure S3.

Article Snippet: The human TBX1 isoform C cDNA clone was purchased from OriGene.

Techniques: Electrophoretic Mobility Shift Assay, Binding Assay, Activity Assay, Mutagenesis, DNA Binding Assay, Transfection, Control, Plasmid Preparation

Journal: Scientific reports

Article Title: A 3 base pair deletion in TBX1 leads to reduced protein expression and transcriptional activity.

doi: 10.1038/srep44165

Figure Lengend Snippet: Figure 5. Proteasome inhibitor MG-132 increases both wild-type and mutant TBX1 protein level. C2C12 cells and 3T3 cells were transfected with equal concentrations of TBX1-encoding plasmids (the wild- type or the mutant 102delK) and treated with 20uM MG-132 or 10uM E-64 for 8 hours. The TBX1 levels were determined by Western blot (A: C2C12 cells; B: 3T3 cells). GAPDH was used as an internal control. n = 3. Wt: wild-type; Mut: the TBX1102delK mutant; vehicle: DMSO. The full-length image is presented in the Supplementary Figure S4.

Article Snippet: The human TBX1 isoform C cDNA clone was purchased from OriGene.

Techniques: Mutagenesis, Transfection, Western Blot, Control