Anti tbk1

We processed western blot analysis to examine the protein levels of TRL4/TRIF signaling pathway downstream in myocardial tissue. Protein extraction was exerted by the RIPA Lysis Buffer and concentration detection was determined using a bicinchoninic acid protein assay kit (both from Beyotime, Jiangsu, China). Equal amounts of protein were separated by 12% sodium dodecyl sulfate-poly-acrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane by electroblotting. The membrane was blocked by 5% defatted milk with Tris-buffered saline Tween-20 (TBST) for 2 h at room temperature in case of nonspecific binding. Then, the rinsed membrane was incubated with the respective specific primary antibody overnight at 4°C, followed by a peroxidase conjugated secondary antibodies. We used anti-TRIF (dilution 1:600), anti-TBK1 (dilution 1:1,000) (both from Santa Cruz Biotechnology, Inc.), anti-IRF3 (dilution 1:1,000) and anti-p-IRF3 (dilution 1:600) (both from Cell Signaling Technology, Danvers, MA, USA). horseradish peroxidase glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was counted as a housekeeping gene to normalize the intensity of different samples. Band was measured with an enhanced chemiluminescence (ECL) reagent (Thermo Fisher Scientific, Waltham, MA, USA) and a analyzed with BandScan 5.0 software.