Plasmids, Antibodies, and Resins for Wnt Signaling

The following plasmids have been described: LDB1-FLAG, SSDP-FLAG (Fiedler et al., 2015 (link)); murine FLAG-B9L, FLAG-ΔC (called FLAG-ΔCter), GFP-B9L (Adachi et al., 2004 (link)); GFP-Lgs, GFP-TCF4 (Townsley et al., 2004 (link)). Human GFP-BCL9 was generated by exchanging the FLAG tag of FLAG-BCL9 (de la Roche et al., 2008 (link)). Mutants were made from parental plasmids using standard site-directed mutagenesis procedures, and confirmed by sequencing. TLE3 was amplified by PCR from 6xMyc-TLE3 (Hanson et al., 2012 (link)) and subcloned in pcDNA3.1-N-HA, and mutants (WD40, ΔWD40) were generated from this subclone.
The following antibodies and resins were used: α-GFP RRID:AB_439690, α-FLAG RRID:AB_262044, α-HA RRID:AB_390918, α-β-actin (for human lysates) RRID:AB_476744 (Sigma-Aldrich, St. Louis MO, USA); α-BCL9 RRID:AB_2063609, α-BCL9-2 RRID:AB_2063747 (Abingdon, UK); α-β-actin (for fly lysates) RRID:AB_2305186, α-BirA RRID:AB_300830, α-Pygo2 RRID:AB_10863482 (Abcam, Cambridge, UK); α-ABC RRID:AB_11127203 (Cell Signaling Technology). Rat α-Lgs antiserum (Eurogentec, Liège, Belgium) was obtained from pre-bleed immunizations with gluthathione S-transferase-purified Lgs_232-555. GFP-Trap resin RRID:AB_2631357 (Chromotek, Planegg, Germany) was used for coIP assays, and α-FLAG M2 affinity gel RRID:AB_10063035 (Sigma-Aldrich) was used for RIME.

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van Tienen L.M., Mieszczanek J., Fiedler M., Rutherford T.J, & Bienz M. (2017). Constitutive scaffolding of multiple Wnt enhanceosome components by Legless/BCL9. eLife, 6, e20882.

Publication 2017

α-GFP α-FLAG α-β-actin α-BirA α-ABC GFP-Trap resin α-FLAG M2 affinity gel

Actin Antibodies Antiserum Assays Bcl9 Gluthathione Human Immunizations Ldb1 Murine Parental Plasmids Resin Site directed mutagenesis procedures Tcf4 Transferase

Corresponding Organization :

Other organizations : MRC Laboratory of Molecular Biology

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Variable analysis

independent variables

Mutants made from parental plasmids using standard site-directed mutagenesis procedures

dependent variables

Protein expression and localization

control variables

Parental plasmids
Sequencing to confirm mutant constructs

controls

Positive control: Parental plasmids
Negative control: Not mentioned

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