Cells were cultured in big dishes until it reached 60–70%, treated with or without NF & SFN for 24 h & 48 h, respectively. Cells were placed on ice for at least 30 min and centrifuged at 12,000 × g for 10 min; afterward, the supernatant was collected and analyzed. Extracted protein concentrations were determined through the BCA Protein Assay Kit (Heart biology technology Ltd. Xian, China). Proteins were separated by 5-12% SDS-PAGE gel electrophoresis and transferred onto PVDF membranes (Merck KGaA, Darmstadt, Germany). The immune-blots were incubated with blocking (5% non-fat milk) solution for 1 h, incubated overnight at 4°C with primary antibodies (1:1000). Then secondary antibodies were diluted at 1:500–1:10,000 followed by the incubation for 60 min at room temperature, and blots were developed by Minichemi® Sagecreation (Baltimore Ave USA). Image J software (National Institutes of Health, Bethesda, MD, USA) was used to analyze and quantify signal intensities. The signals were normalized internal control by GAPDH.

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