Phospho tak1 thr184 187

The following primary antibodies were used for Western blot: antibodies against survivin (#2803), XIAP (#14334), Caspase 8 (#9746S), Caspase 9 (#9508S), Caspase 3 (#9662S), cleaved-Caspase 3 (#9661S), PARP (#9542 L), ubiquitin (#3933S), RIP1 (#3493S), phospho-RIP1 (Ser166) (#44590), RIP3 (#13526S), phospho-RIP3 (Ser227) (#93654), phospho-MLKL (Ser358) (#91689S), TAB1 (#3226), TAK1 (#5206), phospho-TAK1 (Thr184/187) (#4508) and GAPDH (#5174) were from Cell Signaling Technology; anti-Caspase 7 antibody (#sc-28,295) was purchased from Santa Cruz and anti-MLKL antibody (#184718) was purchased from and Abcam. Z-VAD-fmk (Sigma-Aldrich, V116) and Nec-1 s (Biovision, 2535–1) were used to reveal apoptotic and necroptotic cell death, respectively. Cycloheximide (CHX, C7698-5G) was purchased from Sigma-Aldrich. MG132 (S2619) was obtained from Selleckchem.

The mice were euthanized by cervical dislocation, and total protein lysates from the mouse myocardium were prepared as previously described (Lu et al., 2014 (link)). In addition, protein lysates for phospho-TGFβ type 2 receptor (TβR2) and TGFβ type 1 receptor (TβR1) were first prepared by immunoprecipitation with the primary antibody TβR2 (Cell Signaling Technology, USA), TβR1 (Abcam, USA) and Protein A/G PLUS-Agarose (Santa Cruz, USA). After SDS-PAGE and transfer of the bands to nitrocellulose (Millipore, USA), the membranes were incubated overnight with antibodies against tomoregulin-1 (R&D, USA), phospho-serine/threonine kinase (Abcam, USA), TβR2 (Cell Signaling Technology, USA), TβR1 (Abcam, USA), phospho-TAK1 (Thr184/187) (Cell Signaling Technology, USA), phospho-JNK (Thr183/Tyr185) (Cell Signaling Technology, USA), TAK1 (Cell Signaling Technology, USA) and JNK (Cell Signaling Technology, USA). After incubation with the appropriate secondary antibody for 1 h at room temperature, antibody binding was detected with an HRP-conjugated immunoglobulin G (Santa Cruz, USA) using a chemiluminescence detection system (Santa Cruz, USA). The quantitative analysis of the level of proteins were normalized to GAPDH, and the bands were quantified using the ImageJ software.