Cells were transfected with 5µg each of plasmids encoding for DLL4(GeneCopoeia, Rockville, MD, EX-U1091-Lv241) and GFP tagged Ubiquitin {(GFP-Ub); Addgene, 11928}[18 (link)] using GeneJuice (Millipore, Burlington, MA). 24 hours later, siRNA or microRNA were transfected with RNAiMAX. 48 hours later, cells were immuno-precipitated with IgG or anti-GFP and Protein G-agarose (GenDEPOT). Immuno-precipitates were probed for DLL4 by western blot.
Protocol detail
DLL4 Ubiquitination Assay Protocol
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Corresponding Organization : The University of Texas Health Science Center at Houston
Other organizations : Rice University, Neurological Surgery, The Ohio State University Wexner Medical Center, Tri-Institutional PhD Program in Chemical Biology, Cornell University, City Of Hope National Medical Center, University of Pittsburgh
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Variable analysis
independent variables
- Plasmids encoding for DLL4 (GeneCopoeia, Rockville, MD, EX-U1091-Lv241)
- GFP tagged Ubiquitin {(GFP-Ub); Addgene, 11928}
- SiRNA or microRNA
- Immunoprecipitation of DLL4 with IgG or anti-GFP and Protein G-agarose
- Western blot analysis of immunoprecipitated DLL4
- Transfection reagent GeneJuice (Millipore, Burlington, MA)
- Transfection reagent RNAiMAX
- Positive control: Immunoprecipitation with anti-GFP
- Negative control: Immunoprecipitation with IgG
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