Fdr007

Cells (5 × 10⁶ cells) or colon tissue were lysed for 30 min, and lysates were harvested by centrifugation at 13,200 × g for 12 min. Protein concentration was quantified by Pierce™ BCA protein assay kit, and equal amounts of protein were denatured at 100 °C in a loading buffer. Protein samples (~30 μg) were separated on 10% gradient SDS-PAGE gels and transferred to 0.45-μm PVDF membrane (IPVH00010, Millipore, Germany). Membranes were blocked with 5% nonfat milk and then incubated with primary antibodies against the following target proteins: anti-4-HNE (1:1000, ab46545, Abcam, USA), anti-ACSL4 (1:1000, R24265, Zenbio, China), anti-FTH (1:1000, ab65080 Abcam, USA), anti-FTL (1:1000, 10727-1-AP, Proteintech, China), anti-GPX4 (1:1000, ab125066, Abcam, USA), and anti-β-actin (1:3000, ab6276, Abcam, USA). Protein expressions were visualized by incubating the membranes with HRP AffiniPure Goat Anti-Rabbit IgG (H + L) (1:3000, Fude biotech, FDR007) or HRP AffiniPure Goat Anti-Mouse IgG (H + L) (1:3000, Fude biotech, FD0142) and following ECL western blotting substrate (FD8030, Fude Biological Technology, China). The immunoblots were imaged on a Chemiluminescence Image Analysis System (Tanon 5200, Tanon Science and Technology, China).

Proteins were extracted using RIPA buffer (Beyotime, Shanghai, China), and the concentration was determined by a BCA kit (ThermoFisher Scientific). An equivalent amount of proteins was isolated by SDS-PAGE, and transferred to polyvinyl fluoride membrane (Merck KGaA). After incubation with primary antibodies overnight at 4°C, and incubation with horseradish peroxidase-conjugated secondary antibodies (FDM007 and FDR007, Fudebio, Hangzhou, China) for 2 h. The membranes were treated with the enhanced chemiluminescent reagents (MILLIPORE, WBKLS0500). The signals were examined by ChemiDox (bio-rad, USA) with the treatment of an enhanced chemiluminescence kit (FD8030, Fudebio, Hangzhou, China). The primary antibodies involved in the present study were GAPDH (1:1000, Abcam), anti-S1PR2 (1:500, Proteintech), anti-AKT (1:1000, Wanleibio), anti-p-AKT (Ser473) (1:1000, Wanleibio).

Whole-cell extracts were prepared using protein lysis buffer (Invent Biotechnologies, Plymouth, MN, USA). A bicinchoninic acid (BCA) protein assay kit (Fudebio, Hangzhou, China) was used to determine the protein concentrations. Total proteins (30 μg) were separated using 10% SDS-PAGE and electrotransferred to polyvinylidene fluoride membranes. The membranes were blocked in 5% nonfat milk for 1 h at room temperature and then probed with primary antibodies against CMTM2 (ABP53869, Abbkine, Wuhan, China, 1 : 1000), SRY-box transcription factor 2 (SOX2) (11064-1-AP, Proteintech, Rosemont, IL, USA, 1 : 600), NANOG (67255-1-Ig, Proteintech, 1 : 10000), CD44 (15675-1-AP, Proteintech, 1 : 5000), and GAPDH (60004-1-Ig, Proteintech, 1 : 1000) overnight at 4°C. Subsequently, the membranes were incubated with goat anti-rabbit antibodies diluted with 0.3‰ in TBST (FDR007, Fudebio, Guangzhou, China, 1 : 10000) or goat anti-mouse antibody diluted with 0.3‰ TBST for 1 h at room temperature. The membranes were then scanned using the Tanon 5200 automated image analysis system (Tanon Science and Technology Co., Ltd, Shanghai, China), and the ImageJ software (National Institutes of Health) was used to evaluate the band intensity.