Anti snail

Western blotting analysis was conducted as previously described [41 (link)]. The following primary immunoblotting antibodies were used: anti-GAPDH, anti-E-cadherin, anti-Fibronectin, anti-Vimentin, anti-Snail, anti-Slug, anti-Twist, anti-MMP-9, anti-MMP-2, anti-pro caspase-3, and anti-active caspase-3 (Epitomics, Burlingame, USA), anti-HIF-1α and anti-β-catenin (Proteintech, Chicago, USA), anti-N-cadherin (Cell Signaling Technology, Beverly, MA), and poly (ADP-ribose) polymerase (PARP) (Santa Cruz, CA).

Antibodies corresponded to anti-E-cadherin (mouse monoclonal (clone HECD-1); Invitrogen); anti-DSG2 (mouse monoclonal (clone DG3.10); Fitzgerald, Acton, MA); anti-β-actin (mouse monoclonal [clone AC-74]; Sigma-Aldrich, St. Louis, MI); anti-HA-tag (rabbit monoclonal [clone C29F4]; Cell Signaling, Danvers, MA); anti-Snail (rabbit polyclonal; Abcam, Cambridge, MA); anti-pAKT(Ser473) (rabbit monoclonal (clone 736E11); Cell Signaling); and anti-CK8/18 (guinea pig polyclonal; Progen, Heidelberg, Germany).

Cells were rinsed in PBS, fixed for 10 min with 4% formaldehyde in PBS and permeabilized 5 min with 0.1% Triton-X100. After fixation, coverslips were blocked in PBS containing 3% bovine serum albumin for 30 min at 37°C, before being processed for immunofluorescence.
Ki67 immunostaining on tissue slices has been performed following standard procedures.
Antibody dilution was as follows: anti-RhoA-GTP (NewEast Biosciences) 1:400; anti-Aurora B (AbCam ab3609) 1:100; anti-E-Cadherin (BD Biosciences Pharmingen, Bedford, MA) 1:50; anti-Snail (AbCam) 1:50; anti-Ki67 (BD Bioscience Pharmingen, USA; 1:25)
Secondary antibodies conjugated to Alexa-488 or Alexa-594 (Molecular Probes, Invitrogen, San Diego, CA; 1:400) were used as recommended by the supplier. Alexa Fluor 594 Phalloidin (a12381) (Thermo Fisher Scientific Inc., MA, USA) and Alexa Fluor 647–Phalloidin (from Invitrogen, Carlsbad, CA) were utilized for the staining of F-actin.
DNA was counterstained with 0.1 μg/ml 4'-6'-Diamidino-2-phenylindole (DAPI, Sigma-Aldrich, St Louis, MO).
All stained samples were analyzed under a Nikon Microphot-FXA microscope equipped with a CCD camera and digital images were acquired with Nikon NIS-elements software or using a Leica SP5 spectral confocal microscope.

The study protocol was performed according to the principles of the Declaration of Helsinki and the ethics committee of the Affiliated Hospital of Jiangnan University, Wuxi, Jiangsu Province, China. Informed consent was obtained from all patients.
The following primary antibodies were purchased from Abcam (Cambridge, England): anti-E-cad, anti-Vim, anti-Snail, anti-N-cad, anti-Twist, anti-PCNA, anti-Bax, anti-Bcl-2, anti-ZEB1, anti-NEDD9, anti-Myb, and anti-GAPDH. The following primary antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA): anti-t-SRC, anti-p-SRC, anti-t-STAT3, and anti-p-STAT3. PP1, an SRC inhibitor, was purchased from Selleck Chemicals (Houston, TX, USA).

The total protein was extracted from the cultured CFs, and the levels were compared by immunoblotting the proteins based on their expression in the left ventricular peri-infarct area (PIA) of rats [32 (link)]. Antibodies used for probing were anti-FBW7, anti-Snail, anti-SMAD3/4, anti-phosphor-SMAD3/4 (Abcam, Cambridge, UK), anti-Twist, anti-TGF-β, anti-ubiquitin, and anti-actin antibody (Sigma-Aldrich Corp. St. Louis, MO, USA). The corresponding secondary antibodies were purchased from Stressgen Biotechnologies Corporation, Victoria, BC, Canada. Signals were used for detection using the Enhanced Chemiluminescence (ECL) kit (GE Healthcare, UK). Densitometry was employed to quantify the protein levels. β-actin was used as the loading control in the western blotting experiment for normalization.