A11355

Total protein was extracted using RIPA buffer (R0010, Solarbio, Beijing, China). Protein extracts were transferred to PVDF membranes, and then incubated using appropriate antibodies. An ECL imaging system (Tanon-5200, Shanghai, China) was used to detect ECL signals. The primary antibodies used were: anti-LTB4R (ERP7113, 1:1000, Abcam, Carlsbard, CA, USA); anti-GAPDH (A19056, 1:1000, ABclonal, Hubei, China), anti-CDK2 (A0094, 1:1000, ABclonal), anti-CDK4 (A11136, 1:1000, ABclonal), anti-cyclin D1 (A19038, 1:1000, ABclonal), anti-Bcl-2 (A19693, 1:1000, ABclonal), anti-Bax (A19684, 1:1000, ABclonal), anti-caspase3 (9662, 1:1000, Cell Signaling Technology), anti-cleaved-caspase3 (Asp175, 1:1000, Cell Signaling Technology), anti-vimentin (A19607, 1:1000, ABclonal), anti-NCA (A19083, 1:1000, ABclonal), anti-ECA (A18135, 1:1000, ABclonal), anti-AKT (A17909, 1:1000, ABclonal), anti-P-AKT (AP0637, 1:1000, ABclonal), anti-mTOR (A11355, 1:1000, ABclonal), and anti-P-mTOR (AP0115, 1:1000, ABclonal). The second antibody was Anti-Rabbit-IgG(H+L)-HRP (AS030, 1:10,000, ABclonal).

Protein concentrations were determined using a BCA Kit (Beyotime, Beyotime Biotechnology, Shanghai, China). Equal amounts of proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. Antibodies against GPR41 (66811–1-Ig, Sanying, WuHan, China) PI3K (A4992, ABclonal, Wuhan, China), phosphorylated (p)-PI3K (Ab278545, Abcam, UK), Akt (A18675, ABclonal, Wuhan, China), p-Akt (AP1208, ABclonal, Wuhan, China), mTOR (A11355, ABclonal, Wuhan, China), p-mTOR (AP0115, ABclonal, Wuhan, China), RxRα (A19105, ABclonal, Wuhan, China), SREBP1 (A5754, ABclonal, Wuhan, China), PPARγ (A16958, ABclonal, Wuhan, China), St6gal1 (A5754, ABclonal, Wuhan, China), and GAPDH (AB-P-R001, Xianzhi, Hangzhou, China) were used as primary antibodies. All primary antibodies were used at a 1:1000 dilution except for those against p-PI3K (1:500 dilution) and GPR41 (1:2000 dilution). Goat anti-rabbit-HRP (BA1054, Boster Bio, Wuhan, China) and goat anti-mouse-HRP (BA1051, Boster Bio, Wuhan, China) were used as secondary antibodies. The bands were visualized using an Enhanced Chemiluminescence (ECL) Kit (Applygen, Applygen Technologies Co., Ltd., Beijing, China). The protein bands were densitometrically analyzed using the IPP software.

Western blotting was performed as previously described [32 (link)].The antibodies used were as follows: anti-FGF19 (A6589, ABclonal, China, 1:500), anti-TRIM21 (12108–1-AP, Proteintech, China, 1:1000), anti-ANXA2 (sc-28385, Santa Cruz Biotechnology, Texas, USA, 1:1000), anti-Tubulin(FD0064, Fude bio, China, 1:1000), anti-p-PI3K (A0982, ABclonal, China, 1:500), anti-AKT1 (A11016, ABclonal, China, 1:1000), anti-p-AKT1 (AP0140, ABclonal, China, 1:1000), anti-mTOR (A11355, ABclonal, China, 1:500), and anti-p-mTOR (ab109268, Abcam, UK, 1:1000).