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

Journal: Gene Expression

Article Title: Expression of Septin 3 Isoforms in Human Brain

doi:

Figure Lengend Snippet: Upregulation of septin 3 isoforms during RA-induced neural differentiation of SH-SY5Y cells. SH-SY5Y cells were treated for 4 days with vehicle (–) or 2.5 μM RA (+). (a) cDNA synthesized from mRNA of treated cells was used as template for PCR. Three sets of primers were used to detect mRNAs for septin 3A, 3B, and GAPDH as an internal control. The expected sizes of septin 3A (top), 3B (middle), and GAPDH (bottom) mRNAs were 1160, 1308, and 1000 bp, respectively. (b) Lysates from treated cells were separated by 10% SDS-PAGE and then subjected to Western blotting (WB) with antibodies against septin 3A (top), 3B (middle), or β-actin as an internal loading control (bottom).

Article Snippet: The human cDNA fragments of septin 3A, 3B, and Nedd5 were produced by polymerase chain reaction (PCR) from the Human Fetal Brain Large-Insert cDNA library (BD Biosciences Clontech, Palo Alto, CA).

Techniques: Synthesized, SDS Page, Western Blot