ChIP Assay for Foxc1/Foxc2 Binding

For the in vivo ChIP experiments, extracts were prepared from approximate 1 × 10⁷ of P19CL6 cells^{49 (link)} which were transfected by FLAG-tagged Foxc1 or Foxc2 expression vectors 48 hours before harvest. For the ChIP assays, the ChIP-IT Express Chromatin Immunoprecipitation Kits (Active Motif) were used according to manufacturer’s protocol. Primers in PCR reactions were 5′-ACAAGCTTCGCTGGACTGAT-3′ and 5′-TCTCGGCTCACTCTCTGGTT-3′ (−148 + 43). The amplified region corresponded to the mouse Sema3c promoter, which encompasses the FOX-binding site.

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Kodo K., Shibata S., Miyagawa-Tomita S., Ong S.G., Takahashi H., Kume T., Okano H., Matsuoka R, & Yamagishi H. (2017). Regulation of Sema3c and the Interaction between Cardiac Neural Crest and Second Heart Field during Outflow Tract Development. Scientific Reports, 7, 6771.

Publication 2017

ChIP-IT Express Chromatin Immunoprecipitation Kits

Binding site Chip Chromatin immunoprecipitation Mouse Primers Vectors

Corresponding Organization :

Other organizations : Keio University, Tokyo Women's Medical University, Cardiovascular Institute of the South, Stanford University, National Hospital Organization, Northwestern University

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Variable analysis

independent variables

Transfection of P19CL6 cells with FLAG-tagged Foxc1 or Foxc2 expression vectors

dependent variables

Enrichment of the Sema3c promoter region containing the FOX-binding site, as measured by ChIP-PCR

control variables

P19CL6 cell line
Approximate 1 × 10^7 cells
Primers used for PCR amplification of the Sema3c promoter region

controls

Positive control: None mentioned
Negative control: None mentioned

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