Journal: Nucleic Acids Research

Article Title: An optimized split-ubiquitin cDNA-library screening system to identify novel interactors of the human Frizzled 1 receptor

doi: 10.1093/nar/gkm1163

Figure Lengend Snippet: Principle of the yeast R-URA3p-based split-ubiquitin protein–protein interaction system. The interaction of bait and prey proteins leads to the reconstitution of ubiquitin, resulting in proteolytic degradation of the R-URA3p reporter molecule. This renders the yeast cells URA-auxotroph and resistant to 5-FOA. See text for details. UBPs, ubiquitin specific proteases.

Article Snippet: The employed human fetal brain N ub I—split ubiquitin cDNA library was kindly provided by GPC Biotech AG (Munich, Germany) and was constructed as follows: Split-ubiquitin prey expression vectors are based on plasmid PADNS , and are derivatives of PADNX-N ub -BC plasmids as previously described ( ).

Techniques: Ubiquitin Proteomics

Journal: Nucleic Acids Research

Article Title: An optimized split-ubiquitin cDNA-library screening system to identify novel interactors of the human Frizzled 1 receptor

doi: 10.1093/nar/gkm1163

Figure Lengend Snippet: Optimized flowchart for split-ubiquitin protein-protein interaction cDNA-library screens. See text for details.

Article Snippet: The employed human fetal brain N ub I—split ubiquitin cDNA library was kindly provided by GPC Biotech AG (Munich, Germany) and was constructed as follows: Split-ubiquitin prey expression vectors are based on plasmid PADNS , and are derivatives of PADNX-N ub -BC plasmids as previously described ( ).

Techniques: Ubiquitin Proteomics, cDNA Library Assay

Journal: Nucleic Acids Research

Article Title: An optimized split-ubiquitin cDNA-library screening system to identify novel interactors of the human Frizzled 1 receptor

doi: 10.1093/nar/gkm1163

Figure Lengend Snippet: Interaction of human p53 and protein phosphatase 5 (PP5). ( A ) Split-ubiquitin interaction assay. Clones expressing p53 (p53-CRU bait construct) and PP5 (N ub -PP5 prey) were spotted in serial dilutions onto medium lacking uracil (-URA), medium containing 5FOA (+FOA), and control plates selective for the presence of plasmids (C). Interaction is indicated by lack of growth on -URA, paralleled by 5-FOA resistance. A clone co-transformed with p53 and empty prey vector (N ub I) served as negative control. A previously identified bait construct with an inactive URA3p phenocopying a true interaction event was also included. ( B ) GST-pull down experiment. Purified Strep-tagged PP5 (Strep-PP5) expressed from E. coli was incubated with GSH-agarose beads loaded with E. coli expressed GST-p53 fusion protein, or GST alone. Corresponding aliquots of Strep-PP5 ‘input’, ‘unbound’ and ‘bound’ fractions were electrophoresed using SDS-PAGE, and electroblotted to nitrocellulose. Detection was performed first by using a Strep-tactin-HRP conjugate, followed by redetection using an α-GST-HRP conjugate. The asterisk indicates the specific signal.

Article Snippet: The employed human fetal brain N ub I—split ubiquitin cDNA library was kindly provided by GPC Biotech AG (Munich, Germany) and was constructed as follows: Split-ubiquitin prey expression vectors are based on plasmid PADNS , and are derivatives of PADNX-N ub -BC plasmids as previously described ( ).

Techniques: Ubiquitin Proteomics, Clone Assay, Expressing, Construct, Control, Transformation Assay, Plasmid Preparation, Negative Control, Purification, Incubation, SDS Page

Journal: Nucleic Acids Research

Article Title: An optimized split-ubiquitin cDNA-library screening system to identify novel interactors of the human Frizzled 1 receptor

doi: 10.1093/nar/gkm1163

Figure Lengend Snippet: Proteins identified as Frizzled1 interactors in the split-ubiquitin screen

Article Snippet: The employed human fetal brain N ub I—split ubiquitin cDNA library was kindly provided by GPC Biotech AG (Munich, Germany) and was constructed as follows: Split-ubiquitin prey expression vectors are based on plasmid PADNS , and are derivatives of PADNX-N ub -BC plasmids as previously described ( ).

Techniques: Histone Deacetylase Assay