Ab209232

All the kits or reagents for western blotting were obtained from Elabscience (Wuhan, China). Cells were lysed using the RIPA lysis buffer, and the protein concentration was estimated using the BCA Protein Concentration Detection Kit (Catalog No. E-BC-K318). SDS-PAGE (10%; Catalog No. E-IR-R305) was employed to separate proteins, which were then transferred onto PVDF membranes. The membranes were blocked for 1.5 h in 5% skim milk. The membranes were then incubated with primary antibodies (anti-ANP: ab209232, 1:1,000; anti-BNP: ab236101, 1:2,000; anti-β-MHC: ab180779, 1:2,000; and anti-β-actin: ab8227, 1:5,000; Abcam, Cambridge, CA, USA) overnight at 4°C and then incubated with secondary antibodies (Catalog No. E-AB-1003; Elabscience) for 1 h at 25°C. Protein bands were visualized using an ECL luminous detection solution (Catalog No. E-BC-R347). Gray analysis was performed by normalizing to GAPDH levels.

Total protein was extracted from frozen heart tissue using a Minute™ muscle tissue total protein extraction kit (SA-06-MS, Invent Biotechnologies, Inc., United States) following the manufacturer’s instructions. Protein samples were quantified using a BCA Protein Assay Kit (E-BC-K318-M, Elabscience, Wuhan, China). Protein samples were resolved on 10% SDS-PAGE gels (PG212, EpiZyme, Shanghai, China) and transferred onto polyvinylidene fluoride membranes (Millipore, Burlington, MA, United States). The membranes were blocked with 5% fat-free milk and incubated overnight at 4°C with primary antibodies (ANP, 1:1000, ab209232, Abcam, United States; β-MHC, 1:1000, 22280-1-AP, Proteintech, China; p38, 1:1000, ET1702-65, Huabio, China; JNK, 1:1000, RT1550, Huabio, China; ERK1/2, 1:1000, 67170-1-Ig, Proteintech, China; p-p38, 1:1000, ER1903-01, Huabio, China; p-JNK, 1:1000, 4668S, Cell Signaling Technology, USA; p-ERK1/2, 1:1000, 4370T, Cell Signaling Technology, United States; DUSP 6, 1:1000, ET1602-18, Huabio, China; β-ACTIN, 1:1000, ab8226, Abcam, United States). Then, the membranes were washed and incubated with secondary antibodies (Goat anti-Mouse IgG, 1:10000, ab216776, Abcam; Goat Anti-Rabbit IgG, 1:10000, ab6721, Abcam). The protein bands were visualized by ECL (Millipore) and quantified using ImageJ software (National Institutes of Health).