T jnk1 2

Proteins were extracted with cell lysis buffer containing 1% Triton X-100, sonicated using QSonica-Q800R2, quantified using bicinchoninic acid assay (Sigma) and separated on NuPAGE™ 4-12% Bis-Tris precast gels (Thermo Fisher). Immunoblot analyses were conducted according to standard protocol with the following antisera: caspase-3 (#9662), PARP (#9542), β-actin (#3770), IRE1α (#3294), p-eIF2α (#3597), t-eIF2α (#2103), p-P38 MAPK (#4511), t-P38 MAPK (#8690), p-JNK1/2 (#4668), t-JNK1/2 (#9252), p-ERK1/2 (#9101), t-ERK1/2 (#9102), CHOP (#5554), Bim (#2933), Bcl-2 (#4223), Bax (#5023), p-Akt (#4060), t-Akt (#9272), GAPDH (#5174), cyclin b1 (#12231), p-CDK1 substrates (#9477) and FLAG (#14793) from Cell Signaling Technology; p62 (#ab56416) and Mcl-1 (#ab31948) antibodies were purchased from Abcam, while LC3 antibody (#NB100-2220) was obtained from Novus Biologicals. Images were captured using a ChemiDoc™ Touch Imaging System and processed using ImageLab™ Software.

The following antibodies were purchased for use in the western blot experiments: P-JNK1/2 (Cell Signalling; 4668S); t-JNK1/2 (Cell Signalling; 9258P); p-MEK1/2 (Cell Signalling; 9154); t-MEK1/2 (Cell Signalling; 9122); p-ERK1/2 (Cell Signalling; 4370P); t-ERK1/2 (Cell Signalling; 4695P); p-p38 (Cell Signalling; 4511P); t-p38 (Cell Signalling; 8690P); p-IκBαSer32/36 (Cell Signalling; 9246); IκBα (Cell Signalling; 4814); p-p65Ser536 (Cell Signalling; 3033); p65 (Cell Signalling; 4764); GAPDH (Cell Signalling; 2118S); ANP (Santa Cruz Biotechnology; SC-20158); MYH7 (Santa Cruz Biotechnology; SC-53089); MD-1 (Santa Cruz Biotechnology; SC-390613); RP105 (Santa Cruz Biotechnology; SC-27841); TLR4 (Santa Cruz Biotechnology; SC-293072); MD-2 (Novus Biologicals; NB100-56655); Ang II (Sigma; F3165); and U0126 and BAY11-7082 (Selleckchem). Foetal calf serum (FCS) was obtained from Gibco. Cell culture reagents and all other reagents were obtained from Sigma.

Ice‐cold radioimmunoprecipitation assay buffer (containing 50 mmol/L Tris‐Hcl, 150 mmol/L NaCl, 1% Triton X‐100, 1% sodium deoxycholate and 0.1% SDS) was used to extract protein from cardiomyocytes and heart tissue. Then protein was subjected to 10% SDS‐PAGE (50 μg per sample). After transferred onto immobilon membranes (Millipore, Billerica, MA, USA), proteins were incubated with primary antibodies overnight at 4°C. The primary antibodies included the following: ZBTB20 (#ab243143, Abcam, 1:1000 diluted), Bax (#2772), Bcl‐2 (#2870), c‐caspase3 (#9664), T‐caspase3 (#9661), TNFα (#11948), Phospho (P)‐ASK1 (Thr845), total (T)‐ASK1(Thr845)(# #3765), P‐JNK1/2 (#4668p), T‐JNK1/2(#9258), P‐ERK1/2 (#4370P), T‐ERK1/2 (#4695), P‐P38 (#4511P), T‐P38 (#9212P) and GAPDH (#2118, Cell Signaling Technology, 1:1000 diluted), And then incubated with second antibodies of either goat anti‐rabbit IgG (926‐32211; LI‐COR) or goat antimouse IgG (C11026‐03; LI‐COR) for one hour. Analysis and quantification was performed by an Odyssey infrared imaging system (LI‐COR Biosciences). The GAPDH was used as reference.