Ab194533

The proteins were extracted from cells using radio-immunoprecipitation assay lysis buffer (Beyotime, Shanghai, China). The total protein concentrations were detected with bicinchoninic acid protein assay kit (Beyotime, Shanghai, China). The same amount of proteins (20 µg) was packed into each electrophoresis tank, then separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA). Subsequently, the membranes were blocked with 10% skim milk at approximately 25°C. Then, the membranes were washed and incubated with the primary antibodies at 4°C for about 24 hour. Primary antibodies including rabbit anti-CHOP (ab194533, 1:400, Abcam, Cambridge, UK), rabbit anti-Grp78 (ab21685, 1: 500, Abcam), rabbit anti-eIF2α (ab169528, 1: 400, Abcam), rabbit anti-cleaved caspase-3 (ab32042, 1: 400, Abcam), rabbit anti- Janus kinase (JAK)2 (ab108596, 1: 300, Abcam), and rabbit anti-STAT3 (ab68153, 1: 500, Abcam) were used for incubated with goat anti-rabbit IgG H&L (HRP) (ab205718, 1:1000, Abcam) for 1 hour at room temperature. Finally, they were put into an enhanced chemiluminescence system (Bestbio, Shanghai, China) to develop the signals and scan the protein bands with Image J software. GAPDH was used as an endogenous control.

Total proteins were obtained from HCM and AC16 cells with the RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China). Protein quantification was made by the Bradford method. The samples were boiled for 5 min, cooled on ice, and centrifuged for 30s. The supernatant was then taken for polyacrylamide gel electrophoresis. Afterward, the protein was transferred to polyvinylidene fluoride (membranes) under 100 V for 1 hour. Next, the membranes were blocked with 5% skim milk at room temperature (RT) for 1 hour and incubated with the primary antibodies (1:1000) of CHOP (ab194533, Abcam, MA, USA), GRP78 (ab21685), IRE1 (ab37073), p-IRE1 (ab124945), PERK (PA5-15,305, Thermo Fisher, Shanghai, China), p-PERK (PA5-40,294,), anti-LKB1 (ab199970), anti-p-LKB1 (ab63473), anti-AMPK (ab32047), anti-p-AMPK (ab92701), anti-Sirt1 (ab189494), and anti-β-actin (ab8226) overnight at 4°C. After the membranes were cleaned with TBST twice, they were incubated with HRP-labeled secondary antibodies (Abcam) for 1 hour at RT. After being rinsed three times, the membranes were exposed with the ECL chromogenic agent (Millipore, Billerica, MA, USA), and imaged with a membrane scanner.