Chemiluminescent substrate

Protein was harvested using lysis buffer, and a BCA assay was applied to determine the protein concentration. Next, we separated 20 μg of protein using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred protein to a polyvinylidene difluoride (PVDF) membrane (Millipore, MA). After blocking, membranes were incubated with primary antibodies as indicated and with horseradish peroxidase (HRP)-conjugated secondary antibodies. Membranes were developed using a chemiluminescent substrate (Beyotime, China). The antibodies used are listed in Additional file 1: Table S3.

Protein was extracted from the cultured cells using RIPA buffer. A total of 30 μg proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Then, proteins were transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked in TBST containing 5% skimmed milk, and incubated with the primary antibodies including: anti-GRP78 (1:1000; Sigma Aldrich), anti-PERK (1:1000; Sigma Aldrich), anti-p-PERK (1:500; Sigma Aldrich), anti-eIF2α (1:1000; Abcam, Cambridge, UK), anti-p-eIF2α (1:500; Abcam), anti-ATF4 (1:1000; Sigma Aldrich), anti-CHOP (1:500; Sigma Aldrich), anti-SIRT1 (1:1000; CST, Beverly, USA), anti-Bcl-2 (1:1000; Proteintech, Wuhan, China), Bax (1:1000; Proteintech), anti-Mcl-1(1:1000; Sigma), anti-caspase 3 (1:1000; CST), anti-caspase 9 (1:1000; CST), anti-cleaved caspase 3 (1:500; Sigma Aldrich), and anti-β-actin (1:1000; Origene, Rockville, USA), followed by incubation with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies. Finally, chemiluminescent substrate (Beyotime) was used for signal visualization and detection. The density of each band was normalized to its respective loading control (β-actin). The immunoblots were quantified by densitometric analysis.