Western blot analysis was performed on cell lysates collected from the siRNA transfections and the luciferase assay. Twenty micrograms of the cell lysates were run on a 4–15% Criterion TGX Precast gel (Bio-Rad, Hercules, CA) for two hours at 120V. Transferred proteins were probed with both a Fli-1 polyclonal antibody described previously [37 (link)] and an antibody to β-actin (Cell Signaling, Beverly, MA). The results were visualized using the Odyssey Imaging System (LI-COR, Lincoln, NE).
Protocol detail
Western Blot Analysis of Fli-1 and Actin
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Corresponding Organization : Ralph H. Johnson VA Medical Center
Other organizations : Jinan Central Hospital, Shandong University
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Variable analysis
independent variables
- SiRNA transfections
- Fli-1 protein expression
- β-actin protein expression
- Amount of cell lysates (20 micrograms)
- Gel (4–15% Criterion TGX Precast gel)
- Gel running time (2 hours at 120V)
- Antibodies (Fli-1 polyclonal antibody and β-actin antibody)
- Visualization method (Odyssey Imaging System)
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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