Plasmids (Notum wild-type and Notum S239A mutant) are described elsewhere.(11 (link)) Cells were seeded at 70% confluence in 6-well plates and transfected with plasmids (Empty vector, NTM WT and NTM S239A) (2μg) using either Lipofectamine 2000 (Invitrogen) or Mirus TransIT-LT1 (Mirus) for 48 hours and transferred to 24-well plates for further assay. Mouse recombinant Notum protein was synthesized and provided by Indiana Biomedical Research Institute (IBRI).
Protocol detail
Notum Protein Overexpression Assay
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Corresponding Organization :
Other organizations : Indiana University – Purdue University Indianapolis, Boston Children's Hospital, Harvard University, Indiana Hemophilia and Thrombosis Center, Richard L. Roudebush VA Medical Center
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Variable analysis
independent variables
- Plasmid type (Empty vector, NTM WT, NTM S239A)
- Not explicitly mentioned
- Seeding cell density (70% confluence)
- Transfection duration (48 hours)
- Transfection methods (Lipofectamine 2000, Mirus TransIT-LT1)
- Positive control: Mouse recombinant Notum protein synthesized and provided by Indiana Biomedical Research Institute (IBRI)
- Negative control: Empty vector
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