2.5. Western Blot Analysis

Western blotting was used to examine the expression levels of cleaved caspase-3, Bax, Bcl-2, cleaved caspase-9, and cleaved caspase-8 proteins, as well as to evaluate the phosphorylation of GSK-3β, Akt, and PI3K in H9c2 cells, as described previously [14]. To evaluate the expression of cleaved caspase-3 and cleaved caspase-9, the cells were subjected to the following treatments: 2.5 × 10⁻⁴ M labetalol for 24 h; 1% lipid emulsion for 1 h followed by 2.5 × 10⁻⁴ M labetalol for 24 h; inhibitors (10⁻⁶ M LY294002 or 5 × 10⁻⁶ M SB216763) for 1 h followed by 1% lipid emulsion for 1 h and 2.5 × 10⁻⁴ M labetalol for 24 h; 1% lipid emulsion alone for 25 h; or inhibitor alone for 26 h. To assess the expression of cleaved caspase-8, the cells were treated with 2.5 × 10⁻⁴ M labetalol for 6 h. The Bax and Bcl-2 expression levels were determined after the treatment of cells for different durations. The cells were treated with 2.5 × 10⁻⁴ M labetalol for 4 h, 1% lipid emulsion for 1 h followed by 2.5 × 10⁻⁴ M labetalol for 4 h, or 1% lipid emulsion for 5 h. To detect GSK-3β phosphorylation, the cells were subjected to the following treatments: labetalol (2.5 × 10⁻⁴ M) alone for 1 h; 1% lipid emulsion for 1 h followed by labetalol (2.5 × 10⁻⁴ M) for 1 h, SB216763 (5 × 10⁻⁶ M) for 1 h followed by 1% lipid emulsion for 1 h and then labetalol (2.5 × 10⁻⁴ M) for 1 h, 1% lipid emulsion alone for 2 h, or SB216763 (5 × 10⁻⁶ M) alone for 3 h. To evaluate the phosphorylation of Akt and PI3K, the cells were exposed to the following treatments: labetalol (2.5 × 10⁻⁴ M) alone for 1 h; 1% lipid emulsion for 1 h followed by labetalol (2.5 × 10⁻⁴ M) for 1 h; inhibitor (10⁻⁵ M LY294002 or 10⁻⁷ M MK2206) for 1 h followed by 1% lipid emulsion for 1 h and labetalol (2.5 × 10⁻⁴ M) for 1 h; 1% lipid emulsion alone for 2 h; and inhibitor (10⁻⁵ M LY294002 or 10⁻⁷ M MK2206) alone for 3 h [16]. The concentration of LY294002 used in this experiment was chosen by referring to a previous study [16]. Following the treatments, the cells were lysed in radio-immunoprecipitation assay buffer (Cell Signaling Technology) supplemented with protease and phosphatase inhibitor cocktails (Thermo Fisher Scientific, Rockfield, IL, USA). Protein concentrations were determined with a bicinchoninic acid protein assay kit (Thermo Fisher Scientific). The lysate was heated at 100 °C for 10 min to denature the proteins, which were then separated using 8–14% SDS-PAGE and transferred onto PVDF membranes (Millipore, Bedford, MA, USA). The membranes were blocked with tris-buffered saline with 0.5% Tween-20 (TBST) containing 5% skim milk or bovine serum albumin for 1 h at room temperature (22–27 °C) and then incubated with primary antibodies overnight at 4 °C (anti-cleaved caspase-3 [1:1000], anti-cleaved caspase-8 [1:1000], anti-Bax [1:1000], anti-cleaved caspase-9 [1:1000], anti-Bcl-2 [1:250], anti-GSK-3β [1:1000], anti-phospho-GSK-3β [1:1000], anti-Akt [1:1000], anti-phospho-Akt [1:1000], anti-PI3K [1:1000], anti-phospho-PI3K [1:1000], or anti-β-actin [1:10,000]). After washing with TBST, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies against rabbit IgG or mouse IgG (1:5000) for 1 h at room temperature. Protein bands were visualized using the Westernbright™ ECL detection kit (Advansta, Menlo Park, CA, USA), and ECL images were acquired with a ChemiDoc™ Touch Imaging System (Bio-Rad Laboratories Inc., Berkeley, CA, USA). Band densities were determined using the ImageJ software (version 1.45s; National Institutes of Health, Bethesda, MD, USA). The relative density of cleaved caspase-3, Bax, Bcl-2, cleaved caspase-9, and cleaved caspase-8 bands were normalized to β-actin levels. The relative density of bands for the phosphorylated proteins was normalized to the band intensity of the total protein. β-actin served as the loading control. Five independent experiments were performed to determine Bax/Bcl-2, cleaved caspase-9, and cleaved caspase-3 expression. Four independent experiments were performed for determining the expression of cleaved caspase-8 and phosphorylation of GSK-3β and Akt. Six independent experiments were performed to determine PI3K phosphorylation.