Irak1

Fresh myocardial tissue was lysed in western blot lysis buffer containing 1 mM phenylmethanesulfonylfluoride (Beyotime, Shanghai, China). Total proteins were extracted from the lysate and quantified using a bicinchoninic acid protein assay. Polyacrylamide gels (Beyotime) of different concentrations were used based on the molecular weight of proteins to be tested. After samples were loaded and electrophoresed, proteins were transferred to polyvinylidene fluoride membranes using the standard wet transfer method. After blocking with 5% non-fat milk for 2 h, membranes were incubated with primary antibodies in TBST overnight at 4 °C. The following primary antibodies were used: anti-α-SMA (1:10,000, Abcam, Cambridge, UK), IRAK1 (1:1,000, Proteintech, IL, USA), TRAF6 (1:1,000, Abcam), NF-κB (1:1,000, Abcam), p-NF-κB (1:1,000, Abcam), α-SMA (1:1,000, Abcam) and GAPDH (1:10,000, Proteintech). After washing with TBST, membranes were incubated with a secondary antibody at 37 °C for 2 h. Immunoreactive bands were detected by enhanced chemiluminescence ECL (GE, MA, USA) and scanned in the Gel Doc XR system (Azure, C300, CA, USA). Images were analyzed with Image Lab software. Three independent experiments were performed. Averages of all three was analyzed.