Protein extraction kit

Total protein was extracted using a protein extraction kit (Shanghai Yeasen Biotechnology Co., Ltd.) according to the manufacturer's protocol, and the protein concentration was determined using an ultraviolet spectrophotometer (Onedrop1000; Shanghai Genechem Co., Ltd.). A total of 10 µg protein/lane was loaded on a 12% polyacrylamide gel, resolved using SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Sangon Biotech Co., Ltd.). The PVDF membrane was blocked with 5% non-fat milk for 2 h at room temperature. Subsequently, the PVDF membrane was incubated with the corresponding primary antibody against the target protein overnight at 4°C followed by incubation with the secondary antibody at room temperature for 1 h. The protein band was detected using a Beyo ECL Star kit (Beyotime Institute of Biotechnology). The antibodies used were as follows: Anti-PI3K (ab70912; 1:100), anti-MEK1 (ab32091; 1:1,500), anti-ERK1 (ab32537; 1:600), anti-cdc25 (ab111830; 1:2,000) (all Abcam), anti-C-fos (554C1a; 1:500; Santa Cruz Biotechnology, Inc.), anti-ALOX12B (PA5-23608; 1:800; Invitrogen; Thermo Fisher Scientific, Inc.), anti-GAPDH (ab181602; 1:10,000; Abcam), goat anti-mouse IgG antibody (ab97035; 1:2,000) and goat anti-rabbit IgG antibody (ab7090; 1:5,000) (both Abcam).