Wgpvdf45

We used the BCA assay kit to determine the protein concentrations and separated proteins of total extracts on 10% SDS gels (W003-1-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Separated proteins were electrophoretically transferred onto polyvinylidene difluoride membrane (PVDF) (WGPVDF45, Servicebio, Wuhan, China) and blocked with 5% non-fat milk in phosphate-buffered saline (PBS)-Tween for 2 h. The primary antibodies were polyclonal rabbit anti-Bcl-2 (1:1000, GB124830, Servicebio, China), anti-BAX (1:1000, GB12690, Servicebio, China), anti-cleaved-caspase 3 (1:1000, GB11767C, Servicebio, China) and β-actin (1:2000, GB11001, Servicebio, China). They were incubated with the membranes at 4 °C overnight. The membranes were incubated with a secondary antibody conjugated to horseradish peroxidase (Beyotime, Shanghai, China) after being washed with TBST three times for 10 min each time. Bands were visualized using an ECL system (G2014-50ML, Servicebio, China). The ChemiDoc-It2 610 imaging system (UVP, LLC, Upland, CA, USA) was used to conduct the visualization, and Image-Pro Plus 6.0 (Media Cybernetics, Inc., Bethesda, MD, USA) was used for quantification. All experiments were independently replicated three times.

THP-1 were planted in 6-well plates with 2*10⁵ cells per well and PMA induced macrophages for 24 h, then treated with specified concentration of 5% CSE or PBS for 12 h, 24 h, 48 h. The cells were washed with PBS for three times. Collecting cells in each group by trypsin digestion, adding RIPA (Servicebio, G2002-100 ML) lysis buffer containing PMSF (Servicebio, G2008-1 Ml), collecting that supernatant to obtain total protein solution, then protein denaturation. After samples were separated by 12% SDS-PAGE gel electrophoresis, transferred to PVDF membranes (Servicebio, WGPVDF45), blocked with 5% defatted milk for 1 h, the membrane was incubated overnight with primary antibodys (1:1000 diluted GPX4, Servicebio, GB113091; 1:5000 diluted GAPDH, Proteintech, 6004–1) at 4°C, followed by TBST washing three times the next day, incubation with secondary antibody (1:20,000 diluted) for 1 h. Following incubation and washing, uniformly dropwise added that high-sensitivity ECL luminescent liquid (Servicebio, G2014-50 ML). The bands were detected using the Bio-RadChemiDoc MP luminescence imaging system. The images in the film were quantified for the gray value using ImageJ.