Ab7097

The protein expression of Bax, Bcl-2 and caspase-3 was detected by western blot analysis. Five rats from each group were randomly selected and anaesthetized, and the brain tissues were quickly isolated. The hippocampus was rapidly separated on ice and then quickly frozen and stored at −80°C for further use. The hippocampus was homogenized with radioimmunoprecipitation assay lysis buffer. The proteins were then separated by 10% SDS-PAGE and transferred onto PVDF membranes. At room temperature, the membranes were blocked with 5% skim milk for 2 h and then incubated with primary antibodies overnight at 4°C. The antibodies were as follows: caspase-3 (1:2,000, Abcam, ab13847), Bcl-2 (1:1,000, ab196495, Abcam), Bax (1:2,000, Abcam, ab32503), and β-actin (1:2,000, Abcam, ab179467). At room temperature, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1:3,000 diluted, Abcam, ab7097) for 1 h after several washes. We detected the proteins using a chemiluminescence system. Quantity One software (https://download.csdn.net/download/sinat_29367613/8846211) v4.62 was used to quantify the protein bands. All relative optical densities were normalized to β-actin.

Total protein was extracted from the left ventricular myocardium utilizing Total Protein Extraction Kit (Solarbio). Protein samples were separated by 10% sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel electrophoresis and transferred onto the nitrocellulose membranes. The membranes were incubated with the primary antibodies, anti-atrial natriuretic peptide (ANP) (1:1,000 dilution; PA5-29559, Thermo Fisher Scientific, Waltham, MA, USA), anti-β-myosin heavy chain (β-MHC) (1:1,000 dilution; ab37484, Abcam, Cambridge, MA, USA), anti-STIM1 (1:1,000 dilution; MA1-19451, Thermo Fisher Scientific, Waltham, MA, USA), anti-Orai1 (1:1,000 dilution; ab111960, Abcam, Cambridge, MA, USA) at 4 °C overnight after blocking with 5% skimmed milk. The membranes were stained with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG antibody (1:5,000; ab270144, Abcam, Cambridge, MA, USA) or HRP-conjugated anti-rat IgG antibody (1:5,000; ab7097, Abcam, Cambridge, MA, USA) at room temperature. GAPDH antibody (1:5,000; ab8245, Abcam, Cambridge, MA, USA) served as internal control. The bands were developed by enhanced chemiluminescence reagent (Yeasen, Shanghai, China), and the data were analyzed by Image J software. The resulting ratios, expressed as fold change, were used to compare relative protein levels across the samples on blots.