Journal: eLife

Article Title: SAFB regulates hippocampal stem cell fate by targeting Drosha to destabilize Nfib mRNA

doi: 10.7554/eLife.74940

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , pENTR-D-Topo- hTRIM9 (plasmid) , , RRID: Addgene_51032 , pENTR expression vector.

Techniques: Protease Inhibitor, Recombinant, Clone Assay, Bicinchoninic Acid Protein Assay, Mutagenesis, Transfection, Sequencing, esiRNA, Blocking Assay, Plasmid Preparation, Expressing, Software

Journal: Frontiers in Oncology

Article Title: Putative Breast Cancer Driver Mutations in TBX3 Cause Impaired Transcriptional Repression

doi: 10.3389/fonc.2015.00244

Figure Lengend Snippet: Human TBX proteins in alignment with TBX3 . About one-sixth of the T-box, containing the mutations under consideration, is shown (TBX3 from 185 to 216). The complete TBX3 T-box encompasses residues 104–288 . Fully conserved amino acids are marked gray and T-box positions containing similar amino acids are green. In the top line, the three investigated mutations are marked red.

Article Snippet: Construction of cell culture TBX3 expression clones: starting clone TBX3-3xFLAG/pENTR/D-TOPO was constructed by A. Legler based on a TBX3 + 2a cDNA clone (ImaGenes, Berlin, Germany).

Techniques:

Journal: Frontiers in Oncology

Article Title: Putative Breast Cancer Driver Mutations in TBX3 Cause Impaired Transcriptional Repression

doi: 10.3389/fonc.2015.00244

Figure Lengend Snippet: Bacterial expression and DNA binding of mutant TBX3 proteins . (A) GST-TBX3 protein variants after purification by glutathione affinity chromatography. Equal amounts of peak eluate protein were separated on a 10% SDS gel and stained with Coomassie Blue. Normal and point mutant protein fractions yielded a full-length band of 70 kDa [calculated molecular weight of the GST-TBX3(1–342) fusion protein is 65.4 kDa] and a band with ~30 kDa mobility. GST-TBX3(fs163fs2*) migrated with the expected mobility of ~48 kDa. (B) Anti-GST Western blot. A gel as shown in (A) was blotted and probed with anti-GST. Both the 70 and the 30-kDa band reacted with the antibody. Additional bands visible in the Coomassie-stained gel between 65 and 80 kDa were not immunoreactive. (C) Quantification of fusion band expression of the blot shown in (B) . (D) Electrophoretic mobility shift assay of GST-TBX3 protein variants. Equal amounts of fusion protein as determined in (C) were employed. The GST-TBX3(1–342) wildtypic fusion protein effectively shifted the consensus T-box binding element oligonucleotide (arrowhead), the point mutant derivatives T210delT and N212delN were slightly less efficient, H187Y yielded only a faint signal (arrow). (E) Quantification of shift-band intensity. The amount of oligonucleotide shifted by TBX3 was set to 1 ( n = 4). (n = not detected, ns = not significant) (F) EMSA comparison of p.Y163fs2* and the negative control mutant TBX3dm. Control is shift oligonucleotide alone.

Article Snippet: Construction of cell culture TBX3 expression clones: starting clone TBX3-3xFLAG/pENTR/D-TOPO was constructed by A. Legler based on a TBX3 + 2a cDNA clone (ImaGenes, Berlin, Germany).

Techniques: Expressing, Binding Assay, Mutagenesis, Purification, Affinity Chromatography, SDS-Gel, Staining, Molecular Weight, Western Blot, Electrophoretic Mobility Shift Assay, Negative Control

Journal: Frontiers in Oncology

Article Title: Putative Breast Cancer Driver Mutations in TBX3 Cause Impaired Transcriptional Repression

doi: 10.3389/fonc.2015.00244

Figure Lengend Snippet: Repression of p21-Luc reporter by TBX3 and the DNA binding-deficient mutant TBX3dm . (A) Comparison of p21-Luc repression by TBX3 and TBX3dm in transfected COS-7 cells. In all assays, a total of 25 ng of the expression vector was used, with a varying ratio of empty to TBX vector. In the control, 25 ng of empty expression vector was transfected. Repression of the p21-Luc reporter was measured at six different concentrations of TBX3 or TBX3dm. Luc activity in the absence of TBX3 was defined as 0 repression ( n = 3). (B) Comparison of TBX3 and TBX3dm expression. TBX3dm expression is shown in a dilution series of its expression vector. Protein expression was visualized by Western blot of cell extract and FLAG immunodetection. Protein load was controlled with anti-alpha-tubulin. (C) Relative expression of TBX3 and TBX3dm. COS-7 cells were transfected with 25 ng expression vector ( n = 6).

Article Snippet: Construction of cell culture TBX3 expression clones: starting clone TBX3-3xFLAG/pENTR/D-TOPO was constructed by A. Legler based on a TBX3 + 2a cDNA clone (ImaGenes, Berlin, Germany).

Techniques: Binding Assay, Mutagenesis, Transfection, Expressing, Plasmid Preparation, Activity Assay, Western Blot, Immunodetection

Journal: Frontiers in Oncology

Article Title: Putative Breast Cancer Driver Mutations in TBX3 Cause Impaired Transcriptional Repression

doi: 10.3389/fonc.2015.00244

Figure Lengend Snippet: Repression of a p21-Luc reporter by TBX3 and mutant derivatives . (A) The repression of the p21-Luc reporter was measured with 25 ng of expression vector encoding the normal or mutant TBX3 proteins. For control, empty expression vector was transfected. Luc expression is in arbitrary units. The Luc data from six independent experiments were normalized to TBX3 (=1). Average and SD are shown. TBX3dm, Y163fs2*, H187Y, and T210delT were significantly weaker repressors than normal TBX3 but also differed significantly from control. The mutant N212delN did not differ significantly from normal TBX3; hence, was an effective repressor. (B) Quantification of protein expression. Protein expression was visualized by Western blot of cell extract and FLAG immunodetection. Protein load was controlled with anti-alpha-tubulin. (C) Normalization of Luc repression by TBX3 and mutant derivatives to their respective protein concentration. The luciferase activity values of one experiment, for which the protein expression data are shown in (B) , were normalized to the control (=100%). To obtain repression data, these values were subtracted from 100 (0% repression in the control) and shown as black columns (error bars are SD, n = 6 technical replicates). The values after normalization to the different protein levels (see ) are presented by white columns.

Article Snippet: Construction of cell culture TBX3 expression clones: starting clone TBX3-3xFLAG/pENTR/D-TOPO was constructed by A. Legler based on a TBX3 + 2a cDNA clone (ImaGenes, Berlin, Germany).

Techniques: Mutagenesis, Expressing, Plasmid Preparation, Transfection, Western Blot, Immunodetection, Protein Concentration, Luciferase, Activity Assay

Journal: Frontiers in Oncology

Article Title: Putative Breast Cancer Driver Mutations in TBX3 Cause Impaired Transcriptional Repression

doi: 10.3389/fonc.2015.00244

Figure Lengend Snippet: Repression of the endogenous p21 gene by TBX3 and mutant derivatives . COS-7 cells were transfected with 50 ng of expression vector encoding TBX3 or its mutants (the empty vector was transfected in the control experiment). RNA was isolated and the level of p21 transcripts was determined by real-time qPCR. The p21 level is normalized to the control (1.0, black columns, error bars are SDs, n = 3 with three technical replicates each). To correct for differences in the amount of TBX protein, an adjustment was performed as described in Section “ ” (white columns). Only TBX3dm differed significantly in p21 repression from normal TBX3, the three single mutants did not.

Article Snippet: Construction of cell culture TBX3 expression clones: starting clone TBX3-3xFLAG/pENTR/D-TOPO was constructed by A. Legler based on a TBX3 + 2a cDNA clone (ImaGenes, Berlin, Germany).

Techniques: Mutagenesis, Transfection, Expressing, Plasmid Preparation, Isolation

Journal: Frontiers in Oncology

Article Title: Putative Breast Cancer Driver Mutations in TBX3 Cause Impaired Transcriptional Repression

doi: 10.3389/fonc.2015.00244

Figure Lengend Snippet: Protein stability and transcript abundance of TBX3 and mutant derivatives . COS-7 cells were transfected with 50 ng of expression vector encoding TBX3 or its mutants. Twenty-four hours after transfection cycloheximide (CHX) was added to 25 μg/ml. Cells were lysed at the indicated time points. Protein level was quantified by Western blot of cell extract and FLAG bioluminescence immunodetection. Protein load was controlled with anti-alpha-tubulin. (A,B) TBX3 and H187Y, (C,D) TBX3 and N212delN. (E) Quantification of TBX3 transcript levels by real time qPCR. COS-7 cells were transfected with 25 ng of expression vector encoding TBX3 or its mutants (the empty vector was transfected in the control experiment). RNA was isolated 48 h after transfection and analyzed. The data are normalized to TBX3. No TBX3 transcription was detected (nd) in cells transfected with empty vector. The higher level of N212delN RNA relative to normal TBX3 was statistically significant ( n = 3).

Article Snippet: Construction of cell culture TBX3 expression clones: starting clone TBX3-3xFLAG/pENTR/D-TOPO was constructed by A. Legler based on a TBX3 + 2a cDNA clone (ImaGenes, Berlin, Germany).

Techniques: Mutagenesis, Transfection, Expressing, Plasmid Preparation, Western Blot, Immunodetection, Isolation

Journal: Frontiers in Oncology

Article Title: Putative Breast Cancer Driver Mutations in TBX3 Cause Impaired Transcriptional Repression

doi: 10.3389/fonc.2015.00244

Figure Lengend Snippet: Mutations affecting the open reading frame of TBX genes in breast cancer .

Article Snippet: Construction of cell culture TBX3 expression clones: starting clone TBX3-3xFLAG/pENTR/D-TOPO was constructed by A. Legler based on a TBX3 + 2a cDNA clone (ImaGenes, Berlin, Germany).

Techniques:

Journal: Frontiers in Oncology

Article Title: Putative Breast Cancer Driver Mutations in TBX3 Cause Impaired Transcriptional Repression

doi: 10.3389/fonc.2015.00244

Figure Lengend Snippet: Position of TBX3 frameshift mutations in breast cancer . The position of 16 somatic frameshift mutations identified in primary breast cancer tumors is indicated on a schematic drawing of TBX3 [723 amino acids (aa)]. The positions of the T-box, the nuclear localization sequence [290–295 aa, black bar adjacent to T-box ], and the C-terminal repression domain are shown as gray boxes. Fifteen of 16 mutations cluster in the N-terminal half of the protein, most of them in the T-box.

Article Snippet: Construction of cell culture TBX3 expression clones: starting clone TBX3-3xFLAG/pENTR/D-TOPO was constructed by A. Legler based on a TBX3 + 2a cDNA clone (ImaGenes, Berlin, Germany).

Techniques: Sequencing