Following treatment with LBP (50 µg/mL) and/or DDP (10 µM) for 48 hours, MLTC-1 cells were collected and lysed in RIPA buffer (Sigma). The extracted protein concentration was quantified using a BCA Protein Assay Kit (Beyotime, Shanghai, China) and separated on SDS-PAGE gels with 10%Tris-SDS gel. After the electrophoresis, the gel was transferred onto PVDF membranes (Thermo Fisher Scientific). The PVDF membrane was blocked in 5% defatted milk in TBST for 50 minutes and then probed with primary antibodies overnight at 4°C. The following primary antibodies were used: anti-PERK (Cell Signaling; 1:1,000), anti-p-PERK (Cell Signaling; 1:1,000), anti-pelF2α (Cell Signaling; 1:1,000), anti-elF2α (Cell Signaling; 1:1,000), anti-ATF4 (Cell Signaling; 1:1,000), and anti-β-actin (Santa Cruz Biotechnology; 1:1,000), active caspase-3 (Cell Signaling; 1:1,000), active caspase-7 (Cell Signaling; 1:1,000), caspase-12 (Cell Signaling; 1:1,000), LC3 (Cell Signaling; 1:1,000), and Atg 5 (Cell Signaling; 1:1,000). Immunodetection was carried out using anti-rabbit (1:5,000) or anti-mouse (1:5,000) secondary antibodies and enhanced chemiluminescence detection kit (Thermo Fisher Scientific).23 (link)