P fak

Cells were washed with PBS and lysed in Laemmli Sample Buffer (Bio-Rad) and further homogenized with a rotorstator homogenizer. Proteins were isolated and concentrations were determined using the BCATM Protein Assay Kit (Thermo Scientific). 80–120 µg proteins were loaded on a 12–15% sodium dodecyl sulfate-polyacrylamide gel. After electrophoresis, proteins were transferred to a PVDF Western Blotting membrane (Roche). Membrane were blocked with 5% nonfat dried milk (in TBST) for 2 h at room temperature and then incubated overnight at 4°C with Primary antibody (p-FAK, FAK, p-Src, Src, paxillin, vinculin, and talin were purchased from abcam, cleaved-caspase3, caspase3 and β-actin were purchased from Cell Signaling Technology).The membrane was subsequently washed with TBST (5 min×3) and incubated with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology) for 1 h at room temperature. After washing with TBST (5 min×3), bands were detected by enhanced chemiluminescence substrate (Applygen). Band intensities were normalized to its respective internal standard signal intensity. The experiment was repeated 6 times.

Paraffin sections of rat heart were incubated overnight with the anti-angiotensin II type-1 receptor antibody (AT₁R, 1:100 dilution, Proteintech, 25343-1-AP, Chicago, IL, USA), the anti-focal adhesion kinase (phospho Y397) antibody (p-FAK, 1:800 dilution, Abcam, ab81298, Cambridge, UK), anti-focal adhesion kinase antibody (FAK, 1:800 dilution, Abcam, ab40794, Cambridge, UK) and NOX2 rabbit polyclonal antibody (1:400 dilution, Servicebio, Wuhan, China) at 4 °C and subsequently washed with PBS for three times. Then, the sections were incubated with second antibodies includes: IgG-horseradish peroxidase (HRP) (1:100; Dako, Wuhan, China; P0448, Copenhagen, Denmark) and Alexa Fluor-488 goat anti-rabbit antibody (1:500 dilution, Servicebio, Wuhan, China) at room temperature for 1 h. Finally, the sections were incubated with 4′,6-diamidino-2-phenylindole (DAPI). After dying, 3,3′ diaminobenzidine tetrahydrochloride (DAB) horseradish peroxidase Color Development Kit (Hat Biotechnology, Wuhan, China; IS015) was used for chromogenic development. Microphotographs were acquired and analyzed with fluorescence microscopy (ECLIPSE C1, Nikon, Tokyo, Japan). The analysis of the fluorescence area was performed using CaseViewer2.4 software.

Hearts, NRCMs and CFs were lysed using RIPA to extract the total protein and concentration of protein was determined using a BCA Protein Assay kit (TaKaRa). The samples were analysed with 8%‐15% SDS‐PAGE gels and transferred to 0.45‐μm PVDF membranes (Millipore). The membranes were blocked with 5% BSA and incubated with primary antibodies: KCNH2 (ab136467; 1:1000), FOXO3A (ab17026; 1:1000), BCL‐2 (ab59348; 1:1000), p‐AKT (ab81283; 1:1000), AKT (ab179463; 1:1000), p‐FAK(ab81298; 1:1000) and FAK (ab40794; 1:1000) (Abcam); BIM (Cell Signaling Technology, 2933T, 1:1000); PUMA (Santa Cruz Biotechnology, USA,sc‐374223, 1:100); GAPDH (Proteintech, 60004‐1‐lg, 1:1000) was applied as a loading control. The membranes were then incubated with secondary antibodies anti‐mouse (sa00001‐1), anti‐rabbit (sa00001‐2) (Proteintech) and imaged with Amersham Imager 600 or ImageQuant LAS 4000.