Journal: Nucleic Acids Research

Article Title: Intrinsic properties of Tcf1 and Tcf4 splice variants determine cell-type-specific Wnt/β-catenin target gene expression

doi: 10.1093/nar/gks690

Figure Lengend Snippet: Preferential activation of the T/Bra promoter by the hTcf1E and hTcf4E splice variants. ( A ) Western blot analysis of endogenous Tcf/Lef protein levels in nuclear extracts from E14 ESCs, C17.2 and C2C12 cells. GSK3β serves as a loading control. M w , molecular weight standard in kDa. ( B ) Luciferase assay to test the capabilities of different Tcf/Lef family members and splice variants to activate the T/Bra promoter in different cell backgrounds without or with constitutively active β-catenin (βcat). RLA, relative luciferase activity.

Article Snippet: For immunodetection, the following antibodies were used: rabbit anti-Tcf1 (C63D9; Cell Signalling, 1:2000), rabbit anti-Lef1 (C12A5; Cell Signalling, 1:1000), goat anti-Tcf3 (M20; Santa Cruz, 1:1000), rabbit anti-Tcf4 (C9B9; Cell Signalling, 1:1000), mouse anti-β-catenin (no. 610 154; BD Transduction Laboratories, 1:1000), rat anti-HA (3F10; Roche, 1:2000), mouse anti-GSK3β (no. 610 201; BD Transduction Laboratories, 1:1000) and mouse anti-αTubulin (T9026; Sigma-Aldrich, 1:10 000).

Techniques: Activation Assay, Western Blot, Molecular Weight, Luciferase, Activity Assay

Journal: Nucleic Acids Research

Article Title: Intrinsic properties of Tcf1 and Tcf4 splice variants determine cell-type-specific Wnt/β-catenin target gene expression

doi: 10.1093/nar/gks690

Figure Lengend Snippet: Cell-type-specific induction of Wnt/β-catenin target genes. ( A ) quantitative RT-PCR (qRT-PCR) analysis of target gene expression after Wnt3a stimulation in E14 ESCs, C17.2 and C2C12 cells. n.e., not expressed. The values are the expression levels for each gene normalized to Gapdh . ( B ) Assessment of Wnt induction in E14 ESCs, C17.2 and C2C12 cells using luciferase assay. Activation was analysed for Wnt-dependent reporter genes on treatment with Wnt3a or co-transfection of constitutively active β-catenin (βcat). RLA, relative luciferase activity. Unless indicated otherwise, quantitative data in this and all subsequent figures are derived from at least three independent experiments and are the mean values and corresponding standard errors (SEM).

Article Snippet: For immunodetection, the following antibodies were used: rabbit anti-Tcf1 (C63D9; Cell Signalling, 1:2000), rabbit anti-Lef1 (C12A5; Cell Signalling, 1:1000), goat anti-Tcf3 (M20; Santa Cruz, 1:1000), rabbit anti-Tcf4 (C9B9; Cell Signalling, 1:1000), mouse anti-β-catenin (no. 610 154; BD Transduction Laboratories, 1:1000), rat anti-HA (3F10; Roche, 1:2000), mouse anti-GSK3β (no. 610 201; BD Transduction Laboratories, 1:1000) and mouse anti-αTubulin (T9026; Sigma-Aldrich, 1:10 000).

Techniques: Quantitative RT-PCR, Expressing, Luciferase, Activation Assay, Cotransfection, Activity Assay, Derivative Assay

Journal: Nucleic Acids Research

Article Title: Intrinsic properties of Tcf1 and Tcf4 splice variants determine cell-type-specific Wnt/β-catenin target gene expression

doi: 10.1093/nar/gks690

Figure Lengend Snippet: Preferential activation of the T/Bra promoter by the hTcf1E and hTcf4E splice variants. ( A ) Western blot analysis of endogenous Tcf/Lef protein levels in nuclear extracts from E14 ESCs, C17.2 and C2C12 cells. GSK3β serves as a loading control. M w , molecular weight standard in kDa. ( B ) Luciferase assay to test the capabilities of different Tcf/Lef family members and splice variants to activate the T/Bra promoter in different cell backgrounds without or with constitutively active β-catenin (βcat). RLA, relative luciferase activity.

Article Snippet: For immunodetection, the following antibodies were used: rabbit anti-Tcf1 (C63D9; Cell Signalling, 1:2000), rabbit anti-Lef1 (C12A5; Cell Signalling, 1:1000), goat anti-Tcf3 (M20; Santa Cruz, 1:1000), rabbit anti-Tcf4 (C9B9; Cell Signalling, 1:1000), mouse anti-β-catenin (no. 610 154; BD Transduction Laboratories, 1:1000), rat anti-HA (3F10; Roche, 1:2000), mouse anti-GSK3β (no. 610 201; BD Transduction Laboratories, 1:1000) and mouse anti-αTubulin (T9026; Sigma-Aldrich, 1:10 000).

Techniques: Activation Assay, Western Blot, Molecular Weight, Luciferase, Activity Assay

Journal: Nucleic Acids Research

Article Title: Intrinsic properties of Tcf1 and Tcf4 splice variants determine cell-type-specific Wnt/β-catenin target gene expression

doi: 10.1093/nar/gks690

Figure Lengend Snippet: The dependence of endogenous Wnt/β-catenin target gene expression on Tcf1/4 splice variants. ( A ) qRT-PCR showing the effect of a Tcf1/4 single (siTcf1 and siTcf4) or double knockdown (siTcf1/4) on Wnt/β-catenin target gene expression in E14 ESCs. ( B ) qRT-PCR showing the differential capabilities of human Tcf1 (hTcf1) and Tcf4 (hTcf4) splice variants to rescue the Tcf1/4 double knockdown phenotype in E14 ESCs. The values are the expression levels for each gene normalized to Gapdh . ( C ) Schematic representation of the HA-tagged hTcf1 and hTcf4 protein isoforms used for rescue experiments. The locations of the binding domains for β-catenin (βcat), Grg and CtBP co-repressors, the HMG-box, the nuclear localization signal (NLS) and the C-clamp are indicated. ( D ) E14 ESCs were simultaneously transfected with siRNAs and rescue plasmids coding for hTcf1 and hTcf4 protein isoforms as indicated. Subsequent western blot analysis with HA-specific antibodies confirmed similar expression levels for the hTcf1 and hTcf4 splice variants derived from the rescue plasmids. M w , molecular-weight standard in kDa.

Article Snippet: For immunodetection, the following antibodies were used: rabbit anti-Tcf1 (C63D9; Cell Signalling, 1:2000), rabbit anti-Lef1 (C12A5; Cell Signalling, 1:1000), goat anti-Tcf3 (M20; Santa Cruz, 1:1000), rabbit anti-Tcf4 (C9B9; Cell Signalling, 1:1000), mouse anti-β-catenin (no. 610 154; BD Transduction Laboratories, 1:1000), rat anti-HA (3F10; Roche, 1:2000), mouse anti-GSK3β (no. 610 201; BD Transduction Laboratories, 1:1000) and mouse anti-αTubulin (T9026; Sigma-Aldrich, 1:10 000).

Techniques: Expressing, Quantitative RT-PCR, Binding Assay, Transfection, Western Blot, Derivative Assay, Molecular Weight

Journal: Nucleic Acids Research

Article Title: Intrinsic properties of Tcf1 and Tcf4 splice variants determine cell-type-specific Wnt/β-catenin target gene expression

doi: 10.1093/nar/gks690

Figure Lengend Snippet: Tcf1 and Tcf4 are dispensable for maintenance of the active chromatin conformation. ( A ) FAIRE analyses to assess the chromatin conformation at Axin2 , T/Bra , Cdx1 , Cdx2 and Sp5 in E14 ESCs on Tcf1/4 double knockdown with or without concomitant stimulation by Wnt3a. The relative quantities of DNA recovered by FAIRE were determined using qPCR. Analysis of a control region (ctrl) was included to underscore specificity of the results. The data are shown as percent input. The locations of the regions analysed are shown in C. ( B ) qChIP with antibodies specific for H3K4me3 and H3K27me3 to determine association with the promoter regions in Axin2 , T/Bra , Cdx1 , Cdx2 and Sp5 in E14 ESCs on Tcf1/4 double knockdown or knockdown of β-catenin. The data are shown as the fold enrichment over Gapdh .

Article Snippet: For immunodetection, the following antibodies were used: rabbit anti-Tcf1 (C63D9; Cell Signalling, 1:2000), rabbit anti-Lef1 (C12A5; Cell Signalling, 1:1000), goat anti-Tcf3 (M20; Santa Cruz, 1:1000), rabbit anti-Tcf4 (C9B9; Cell Signalling, 1:1000), mouse anti-β-catenin (no. 610 154; BD Transduction Laboratories, 1:1000), rat anti-HA (3F10; Roche, 1:2000), mouse anti-GSK3β (no. 610 201; BD Transduction Laboratories, 1:1000) and mouse anti-αTubulin (T9026; Sigma-Aldrich, 1:10 000).

Techniques: