P erk

We used Ishikawa and SNGM cells during the logarithmic growth period to extract total proteins, and protein content determined by BCA Protein Quantification Kit. Take a protein sample and heat it at 100 °C for 3 min to make the protein fully denatured. After separating the mixed protein sample by using polypropylene gel (10% separation gel, 5% concentrated gel), it was placed onto a polyvinylidene difluoride (PVDF) membrane and sealed with 5% skim milk powder (2.5 g non-fat powdered milk + 50 ml Tris-buffered saline with Tween 20). Incubate these membranes with antibodies at 4 °C for 12–16 h: KIF23 (1:2000; Affinity), p-ERK (1:1000; Beyotime), ERK (1:1000; Beyotime), p-AKT (1:1000; ABclonal), AKT (1:5000; ABclonal), p-PI3K (1:1000; ABclonal), PI3K (1:2000; Bioworld), BCL-2 (1:1000; Beyotime), BAX (1:1000; Beyotime), Caspase-3 (1:1000; Abmart), and GAPDH (1:1000; Beyotime). Subsequently, the sample was coupled with peroxidase-coupled anti-rabbit IgG antibody (1:5000; Beyotime, China) for 2 h under room temperature. ECL enhanced chemiluminescence kit (BOSTER, USA) Using a supersensitive ECL chemiluminescence ready-to-use substrate (BOSTER, USA) to visualize the strip on the Bio-RAD machine.

The total protein was extracted from the corpus callosum of rats for western blot analysis. Briefly, rats were deeply anesthetized with pentobarbital and perfused transcardially with 4°C saline (50 ml). The corpus callosum was rapidly separated and removed on ice. After adding the lysis buffer, the brain tissue was fragmented using a tissue grinder. Protein concentrations of all extracted samples were measured using Bio-Rad Protein Assay (BioRad, Hercules, CA, United States) and bovine serum albumin (BSA) standards. A 30 μg protein sample was loaded and separated by sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane, which was then incubated overnight at 4°C with the following primary antibody: ERK (1:1,000, Cell Signaling Technology, Boston, MA, United States), P-ERK (1:1,000, Beyotime, China), JNK (1:1,000, Cell Signaling Technology, Boston, MA, United States), P-JNK (1:1,000, Beyotime, China), P38 (1:1,000, Cell Signaling Technology, Boston, MA, United States), P-P38 (1:1,000, Beyotime, China) and GAPDH (1:1,000, Servicebio, Wuhan, China). Next, the membrane was washed and incubated with horse-radish peroxidase-labeled goat anti-rabbit and goat anti-mouse secondary antibody (1:10,000, Boster, China) for 1 h at room temperature. Finally, the enhanced chemiluminescence system was used to observe protein bands.