Gapdh

Extract total proteins from cells, and then the protein content was evaluated. The protein was separated by electrophoresis on a 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred to a Hybond-P polyvinylidene difluoride (PVDF) membrane. The membrane was blocked in 5% nonfat milk and incubated with Notch-1, Hes-1, Jagged-1, E-cadherin, and vimentin antibodies (1:1,000, CST, Danvers, MA, USA) 4 °C for the night. Then, incubation with the horseradish peroxidase (HRP)-conjugated secondary antibody (1:2,000, CST, Danvers, MA, USA) and leave them at room temperature for 1 hour. After washing with PBS, the bound primary antibody was visualized with the Enhanced Chemiluminescence System (Amersham, Piscataway, NJ, USA) and exposed to film. -The same approach was applied to GAPDH (Solarbio, San Diego, CA, USA) for loading control. The relative density was analyzed with the Image J software.

Left atrial tissue was homogenized in lysis buffer containing radioimmunoprecipitation assay (RIPA) buffer and inhibitors of proteases. Protein concentrations were determined by the bicinchoninic acid (BCA) assay. Proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), with 8% gels used for endothelial nitric oxide synthase (eNOS) and Collagen I and 10% gels used for TGF-β1, Smad7, and α-SMA. The fractionated proteins were electrophoretically transferred to nitrocellulose membranes (Amersham, Piscataway, NJ, USA). The membranes were incubated with the appropriate primary antibodies for each target protein at 4°C overnight. The primary antibodies for TGF-β1 (1: 500), eNOS (1: 500), Collagen I (1: 500), and α-SMA (1: 200) were from Abcam (Cambridge, MA, USA). The Smad7 (1: 1000) primary antibody was from Aviva Systems Biology (San Diego, CA, USA). The secondary antibodies for Smad7 and eNOS were horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Abcam), diluted 1: 5000. The secondary antibodies for TGF-β1, Collagen I, and α-SMA were HRP-conjugated goat anti-mouse IgG (Abcam), diluted 1:5000. Stained bands were visualized after incubation with HRP-conjugated secondary antibody using enhanced chemiluminescence (ECL) detection reagents (Amersham). GAPDH (1: 2500; Solarbio, Beijing, China) was used as a loading control.

The radioimmunoprecipitation assay buffer (Thermo Fisher Scientific) was used to isolate the total protein. Following quantification with bicinchoninic acid kit (Thermo Fisher Scientific), 20 μg samples were separated via sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred on the polyvinylidene fluoride membrane (Bio-Rad, Hercules, CA, USA). The membranes were blocked in 5% fat-free milk for 1 h and then incubated with primary antibodies (E-cadherin [ab40772, 1:8000; Abcam, Cambridge, UK], intercellular adhesion molecule-1 [ICAM-1] [ab109361, 1:1000; Abcam], vitronectin [ab45139, 1:2000; Abcam], proliferating cell nuclear antigen [PCNA] [ab18197, 1:5000; Abcam], matrix metalloproteinase 9 [MMP9] [ab76003, 1:5000; Abcam], AQP4 [ab81355, 1:300; Abcam], and GAPDH [1:5000; Abcam]) overnight, followed by interacting with secondary antibodies (ab6721, 1:2000; Abcam) for 2 h. Then blots were exposed to enhanced chemiluminescence (Solarbio) and detected via ImageJ v1.8, with GAPDH as a normalized control.