T tak1

Total proteins were extracted from heart tissues of TAC mice, cardiac fibroblasts, cardiomyocytes, and concentrated supernatants of the culture medium, and quantified using the BCA protein assay kit (Thermo Scientific, USA). According to the molecular weight of target proteins, the protein samples were separated using SDS/PAGE (10% or 15% gel), then transferred to Immobilon-P PVDF membranes. After blocking with western blocking buffer (5% BSA: bovine serum albumin 2.5 g + TBST 50 mL), the membranes were incubated overnight at 4 °C with the following primary antibodies: CysC (1:1,000, ab109508, Abcam, Cambridge, MA, USA), phosphorylated extracellular-regulated protein kinase (pERK, 1:5,000, #4370), tERK (1:5,000, #4695), pP38 (1:1,000, #4511), tP38 (1:1,000, #8690), pJNK (1:1,000, #4668), tJNK (1:1,000, #9252), pTAK1 (1:1,000, #4508), tTAK1 (1:1,000, #5206), and GAPDH (1:10,000, #8884, all Cell Signaling Technology, Danvers, MA, USA). Membranes were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:1,000) for 1 hour at room temperature. After treatment with Pro-Light chemiluminescent detection kit (Tiangen Biotech Inc., Beijing, China), the proteins were detected using Omega Lum C imaging system (Aplegen, CA, USA).