Enhanced chemiluminescence system

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.