Antimouse or anti rabbit igg secondary antibodies

Protein lysates were extracted from atria, and the concentration was determined using a bicinchoninic acid (BCA) protein assay. Proteins (20–40 μg) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane, which was incubated with primary antibodies against collagen-1 (1:1000; Abcam, Cambridge, UK), collagen-3 (1:1000; Abcam), α-SMA (1:3000; Abcam), MMP9 (1:3000; Abcam), p-AKT (1:1000; Cell Signaling Technology, Danvers, MA, USA), AKT (1:3000; Cell Signaling Technology), p-mTOR (1:3000; Cell Signaling Technology), mTOR (1:3000; Cell Signaling Technology), p-ERK1/2 (1:3000; Cell Signaling Technology), ERK1/2 (1:3000; Cell Signaling Technology) and GAPDH (1:3000; Cell Signaling Technology). The membranes were then incubated with antimouse or anti-rabbit IgG secondary antibodies (1:5000; Cell Signaling Technology) at room temperature for 1.5 h. Bands were visualized using a Tanon 4800 chemiluminescence detection system (Tanon, Shanghai, China). All blots were analyzed by using the ImageJ (Bethesda) and normalized to GAPDH levels.

Protein lysates were extracted from left atria, and the concentration was determined using a BCA protein assay. Proteins (40‐60 μg) were subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (Bio‐Rad), which was incubated with primary antibodies against CXCR2, TGF‐β1, Smad2/3, phosphorylated (p)‐Smad2/3, P65, p‐P65, NOX1 and NOX2 (Cell Signaling Technologies, Boston, MA, USA), and GAPDH (Proteintech Group. Inc, Rosemont, IL, USA).¹⁴, ¹⁷ Antimouse or anti‐rabbit IgG secondary antibodies were purchased from Cell Signaling Technologies. All blots were analysed by using the ImageJ and normalized to GAPDH levels.