A ChIP assay kit from Millipore (#17–371) was used according to the manufacturer’s protocol. Briefly, Hela cells were transfected with pPGS-3HA-TEAD1 (Addgene #33055)33 (link) and/or pcDNA3.1(−)-Malat1. After crosslinking, 5 μg of the antibody against HA (Abcam, #ab9110), YAP (Cell Signaling Technology, #14074), or normal rabbit IgG (Santa Cruz Biotechnology, #sc-2027) was added to immunoprecipitate HA-tagged TEAD1 or endogenous YAP. For MALAT1 knockout MDA-MB-231 and Malat1-overexpressing LM2 cells, 5 μg of the antibody against TEAD1 (BD Biosciences, #610922), YAP (Cell Signaling Technology, #14074), normal mouse IgG (Santa Cruz Biotechnology, #sc-2025), or normal rabbit IgG (Santa Cruz Biotechnology, #sc-2027) was added to immunoprecipitate endogenous TEAD1 or endogenous YAP. After immunoprecipitation, protein-DNA crosslinks were reversed and DNA was purified to remove the chromatin proteins and used for qPCR. Primers specific for known YAP-TEAD target gene promoters (ANKRD1, CTGF, and CYR61) were from a previous study40 (link). Primers specific for ITGB4 and VEGFA promoters were designed in this study. Primer sequences are listed in Supplementary Table 2 . The results are presented as fold enrichment (normalized to IgG).
Protocol detail
ChIP-qPCR Assay for YAP-TEAD Targets
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Antibody
Cells
Chip assay
Chromatin
Ctgf
Gene
Hela cells
Immunoprecipitation
Itgb4
Mouse
Primer
Protein dna
Proteins
Rabbit
Corresponding Organization :
Other organizations : The University of Texas MD Anderson Cancer Center, Baylor College of Medicine, Hokkaido University, The University of Texas Health Science Center at Houston
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Variable analysis
independent variables
- Transfection of Hela cells with pPGS-3HA-TEAD1 and/or pcDNA3.1(-)-Malat1
- MALAT1 knockout in MDA-MB-231 cells and MALAT1 overexpression in LM2 cells
- Enrichment of known YAP-TEAD target gene promoters (ANKRD1, CTGF, CYR61, ITGB4, VEGFA) as measured by qPCR
- ChIP assay kit from Millipore (#17–371) used according to the manufacturer's protocol
- Antibodies used for immunoprecipitation: HA (Abcam, #ab9110), YAP (Cell Signaling Technology, #14074), normal rabbit IgG (Santa Cruz Biotechnology, #sc-2027), TEAD1 (BD Biosciences, #610922), normal mouse IgG (Santa Cruz Biotechnology, #sc-2025)
- Positive control: Immunoprecipitation of HA-tagged TEAD1 and endogenous YAP
- Negative control: Immunoprecipitation with normal rabbit IgG
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