P tak1 thr184 187

Tissue or cell proteins were prepared according to published protocols (Liao et al., 2015 (link); Yuan et al., 2016 (link)). Briefly, mouse heart tissue or NRCMs were lysed in RIPA lysis buffer for total protein extraction. BCA Protein Assay Kit was used for quantifying protein concentration. Proteins (50 μg/sample) from different samples were used for SDS-PAGE electrophoresis and subsequently transferred to PVDF membrane (Millipore). After blocking with 5% BSA for 1 h, blots were then incubated with primary antibodies overnight at 4°C. The next day, these blots were incubated with secondary antibody conjugated with peroxidase (1:10,000, the Jackson Laboratory) for 1 h at room temperature. All blots were visualized using ChemiDocTM XRS+ (Bio-Rad). The bands were quantified and analyzed using Image-Pro Plus 6.0. The expression of specific proteins was normalized to corresponding GAPDH before relative quantitative analysis. The primary antibodies used in this study were purchased from Cell Signaling Technology (CST): GAPDH (#2118, 1:1,000), p-ERK1/2^{Thr202/Tyr204} (#4370, 1:1,000), T-ERK1/2 (# 4695, 1:1,000), p-p38^{Thr180/Tyr182} (#4511, 1:1,000), T-p38 (#9212, 1:1,000), p-JNK^{Thr183/Tyr185} (#4668, 1:500), T-JNK (#9258, 1:1,000), RIP2 (#4142, 1:1,000), p-TAK1^Thr184/187 (#4508, 1:500), T-TAK1 (#5206, 1:500), TGF-β, p-smad3^ser423/425 (#8769, 1:500), and T-smad3 (#9513, 1:1,000).