Immunoprecipitation of Snail1, pan-Kla, pan-Kac, CBP, and p300

Immunoprecipitation was performed as described in our previous studies (6 (link), 58 (link)). Briefly, about 200 μg of protein of HUVECs, heart tissue, or cardiac endothelial cells was incubated with 2 μg of anti-Snail1, anti–pan-Kla, anti–pan-Kac, anti-CBP, or anti-p300 antibody overnight at 4°C followed by adding 20 μl of protein A/G agarose beads (Santa Cruz Biotechnology) and incubating for another 4 hours. We then washed the precipitates and boiled them in SDS sample buffer. The supernatant was subjected to immunoblotting with anti–pan-Kla antibody, anti–pan-Kac antibody (1:1000 dilution; ABclonal, A2391), anti-CBP antibody (1:1000 dilution; Cell Signaling Technology, 7389S), anti-p300 antibody (1:1000 dilution; Cell Signaling Technology, 86377S), or anti-Snail1 antibody (1:500 dilution; eBioscience, 14-9859-82), respectively.