ChIP Assay for Transcription Factor Binding
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Corresponding Organization : West China Hospital of Sichuan University
Other organizations : University of North Carolina at Chapel Hill
Variable analysis
- ChIP assay performed as previously reported (42 (link))
- Primary HSCs cross-linked by 1.42% formaldehyde followed by quenching with 125 mM glycine
- Cells lysed by ChIP buffer with protease inhibitor cocktail
- Sheared chromatin supernatants added with normal rabbit immunoglobulin G (IgG) (2729, Cell Signaling Technology), anti-Stat5 antibody (94205, Cell Signaling Technology), or anti-Smad3 antibody (ab28379, Abcam)
- Binding of Stat5 and Smad3 to gene promoters including Acta2, Col1a1, Glrx, Bcl2, Il10, and Cdkal1 (measured by real-time PCR using the specified ChIP primers)
- Protein A Magnetic Beads (S1425S, New England Biolabs) used for precipitation
- Normal rabbit immunoglobulin G (IgG) (2729, Cell Signaling Technology) as a negative control
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