Protein extraction reagent kit

A protein extraction reagent kit (Beyotime, Shanghai, China) was utilized to extract total protein from human CCA tissues and cells according to the manufacturer's instructions. Western blotting was done with a modified version of a previous method 17 (link). Anti-SOCS3 antibody was purchased from ABCAM. The NIH Image J software (National Institutes of Health, Bethesda, MD) was used to make the results visualized. GAPDH was used as an internal loading control.

The protein extraction reagent kit (Beyotime, China) and 0.2% phenylmethanesulfonyl fluoride were used to obtain the proteins of cells and tissue samples. Western blotting was done with a modified version of a previous method^{25 (link)}. Anti-BUB1B (1:1000, ab183496), anti-p-c-Jun (1:1000, ab32385), and anti-p-c-Jun N-terminal kinase (JNK) (1:1000, ab4821) were purchased from Abcam. The NIH ImageJ software (National Institutes of Health, Bethesda, MD) was used to make the results visible. GAPDH and α-tubulin were used as an internal loading control.

Neutrophils were seeded into the 6-well plate and treated with NaF (0.25, 0.5, and 1 mM) for 2 h. Previous studies showed that these proteins are still phosphorylated when neutrophils were stimulated by toxins, such as Ochratoxin A, Fumonisin B1, and sodium arsenic [20 (link)–22 (link)]. Meanwhile, NET formation was detected 2 h after NaF stimulation. Therefore, we chose 2 h based on these previous published articles. Total protein was extracted using the Protein Extraction Reagent Kit (Beyotime Biotechnology, China). The protein concentration was measured by the BCA protein quantitative kit (Beyotime Biotechnology, China). The extracted samples were separated by 12% SDS-PAGE and transferred to the PVDF membrane. The membrane was sealed by 5% BSA at room temperature for 2 h. Then, the membrane was incubated with primary antibodies: p38 (Cell Signaling Technology, Cat# 8690), p-p38 (Cell Signaling Technology, Cat# 9215), p-AMPK (Beyotime, Cat#AA393), AMPK (Proteintech, Cat#10929-2-AP), and β-actin (Cell Signaling Technology, Cat# 8457) at 4°C overnight and the second antibody (1 : 10000, ImmunoWay, RS0001, RS0002) at room temperature for 2 h. The reactivity of the primary antibodies used in this study is against Cyprinus carpio. The ECL Plus Western Blotting detection system was used to detect the signal. Finally, the gray level was analyzed by ImageJ software.