Anti snail1 antibody

For in vivo studies, paraffin-embedded heart tissue sections were deparaffinized and rehydrated, blocked in 10% bovine serum albumin (BSA) (Thermo Fisher Scientific, MA, USA), and incubated with primary antibodies, specific anti-GFP antibody (1:100 dilution; ABclonal, AE011), anti-FSP1 antibody (1:50 dilution; Abcam, ab93283), anti-Snail1 antibody (1:100 dilution; eBioscience, 14-9859-82), and anti-MCT1 antibody (1:100 dilution; ABclonal, A9061) at 4°C overnight. For in vitro studies, endothelial cells were fixed with 3.7% formaldehyde (Sigma-Aldrich, MO, USA), permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, MO, USA) in phosphate-buffered saline (PBS), and blocked with 3% BSA in PBS followed by incubating with primary antibodies at 4°C overnight. The following primary antibodies were used for immunofluorescent staining: anti–VE-cadherin antibody (1:50 dilution; Abcam, ab33168), anti-CD31 antibody (1:50 dilution; Abcam, ab28364), anti-FSP1 antibody (1:50 dilution; Abcam, ab93283), and anti-Snail1 antibody (1:100 dilution; eBioscience, 14-9859-82). The next day, slides or cells were stained with secondary antibodies at room temperature for 90 min and mounted in mounting medium with DAPI (Vector Laboratories, CA, USA).

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.