Ab181242

The cell lysate was obtained by employing RIPA lysis buffer (Solarbio; R1200) supplemented with a protease inhibitor cocktail set (Solarbio; A8260). The protein content was subsequently assessed using a bicinchoninic acid assay kit (Thermo Fisher Scientific; 23,227). To separate proteins, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was employed. The proteins were then transferred from the gel to polyvinylidene fluoride (PVDF) membranes (Roche, Basel, Switzerland; 03010040001) using a current of 200 mA for 50 to 60 min, based on protein size. Following this, the membranes underwent incubation with primary antibodies, specifically anti-ANP (1:2000; Abcam; ab181242), anti-BNP (1:2000; Abcam; ab309127), anti-MMP-1 (1:2000; Abcam; ab52631), anti-cAMP (1:2000; Abcam; ab76238), anti-PKA (1:2000; Abcam; ab32514), anti-PKC (1:2000; Abcam; ab32376), anti-PLB (1:2000; Abcam; ab85146), anti-p-PLB (1:500; Abcam; ab62170), anti-CAMKII (1:2000; Abcam; ab134041), p-CaMKII (1:500; Thermo Fisher, PA5-37833), and anti-GAPDH (Abcam; ab8245) overnight at 4 °C. After two washes with TBST, the membranes were incubated with HRP-labeled secondary antibodies for 1 h at room temperature. Subsequently, immunoreactive bands were visualized using an ECL reagent (Amersham, Little Chalfont, UK; RPN2232).

Myocardial tissue was homogenized in lysis buffer containing PMSF and protein kinase inhibitor. After vortexing, the lysate was centrifuged at 13500 rpm/min for 15 min at 4°C and SDS-PAGE was performed. The protein samples separated by SDS-PAGE were then transferred to polyvinylidene fluoride (PVDF) membrane. The membrane was sealed with 5% BSA for 2 h at room temperature and incubated with anti-ERK1/2 (Cell Signaling Technology, #9926, 1 : 1000), anti-JNK (Cell Signaling Technology, #9926, 1 : 1000), anti-p38 MAPK (Cell Signaling Technology, #9926, 1 : 1000), anti-p-ERK1/2 (Cell Signaling Technology, #9910, 1 : 1000), anti-p-JNK (Cell Signaling Technology, #9910, 1 : 1000), anti-p-p38 MAPK (Cell Signaling Technology, #9910, 1 : 1000), anti-ANP (Abcam, ab181242, 1 : 10000), anti-BNP (Abcam, ab236101, 1 : 1000), and anti-GAPDH (Proteintech, 10494-1-AP, 1 : 10000) antibody overnight at 4°C. Goat anti-rabbit IgG (Cell Signaling Technology, #9910, 1 : 1000) was used to block for 2 h, and the membrane was washed three times. ECL was used to visualize these bands. The ImageJ software (http://rsb.info.nih.gov/ij/) was used for densitometry analysis.