P yap

Deparaffinized and rehydrated liver tissue sections were immersed in citrate buffer for antigen retrieval and then incubated with 3% hydrogen peroxide for 10 min to quench endogenous peroxidase activity. Next, sections were blocked with 10% goat serum albumin and incubated with a primary antibody targeting alpha-smooth muscle actin (α-SMA, 1:100; Abways, Shanghai, China), interleukin-1 beta (IL-1β, 1:100; ABclonal, Wuhan, China), YAP (1:100; Santa Cruz Biotechnology, Inc., California, USA), P-YAP (1:100; Abways, Shanghai, China), glutathione peroxidase 4（GPX4, 1:100; Abways, Shanghai, China), solute carrier family 7 members 11 (SLC7A11, 1:250; Proteintech, Wuhan, China), receptor-interacting serine-threonine kinase 3 (RIPK3, 1:100; Signalway antibody, College Park, USA), or caspase8 (1:200; Abcam, Cambridge, UK) at 4 °C overnight. After washing with phosphate-buffered saline (PBS) three times, sections were incubated with the corresponding horse radish peroxidase (HRP)-conjugated secondary antibody at room temperature for 30 min. After washing, sections were visualized with 3,3′-diaminobenzidine (DAB) and stained with hematoxylin. Images were captured and integral optical density (IOD) was determined using Image-Pro Plus 6.0 software. A total of ten non-overlapping fields were analyzed from each specimen.

Collected liver tissues were lysed in radioimmunoprecipitation assay buffer with 1 mM phenylmethylsulfonyl fluoride and phosphatase inhibitors. After the protein concentrations were quantified, an equal amount of total protein (100 μg) was separated by SDS-PAGE and transferred onto PVDF membranes (Millipore, Mass, USA). Membranes were blocked and incubated with primary antibodies, including YAP (1:8000; Proteintech, Wuhan, China), transcriptional co-activator with PDZ-binding motif (TAZ, 1:1000; Bioworld, Minnesota, USA), P-YAP (1:10000; Abways, Shanghai, China), GPX4 (1:1500; Abways, Shanghai, China), SCL7A11 (1:1500; Abways, Shanghai, China), ACSL4 (1:1500; Abways, Shanghai, China), RIPK3(1:1500; Signalway antibody, College Park, USA), or MLKL (1:1500; Signalway antibody, College Park, USA) at 4 °C overnight. Membranes were rinsed with TBS-T, and then incubated with a secondary antibody (anti-rabbit-IR680 conjugates; 1:10000; LI-COR, USA) for 1 h. Finally, protein bands were visualized using the Li-COR Odyssey infrared imaging system and quantified using Image J software.