Anti vasp

A total protein extraction kit (Millipore, Billerica, MA, United States) was used to obtain total protein from atrial tissues. BCA working solution (Thermo Fisher Scientific, MA, United States) was used to determine protein concentrations. Bromophenol blue was added, and the samples were boiled to denature the protein. Proteins were then separated by SDS-PAGE gel electrophoresis and transferred to polyvinylidene fluoride membranes. Membranes were blocked with 5% skim milk powder for 1.5 h and then incubated overnight at 4°C with the following primary antibodies: anti-NPR-A [1:1000], anti-VASP [1:1000], anti-p-VASP [(Ser 239) 1:1000], anti-Akt [1:1000], anti-p-Akt [(Thr 308) 1:1000], anti-GSK-3β [1:1000], anti-p-GSK-3β [ (Ser 9) 1:1000], and anti-β-actin [1:5000]; all from Santa Cruz Biotechnology, Santa Cruz (CA, United States). The membranes were then washed with TBST and incubated with HRP-secondary antibodies at room temperature for 1.5 h. Finally, images were acquired using enhanced chemiluminescence solution.