A11016

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).

By adding RIPA cell lysate containing protease inhibitor, total protein was extracted, vortexed evenly, cultured on ice for 20 min, and the protein concentration was determined by a BCA method. After an addition of 5× loading buffer and mixing well, the protein was boiled at 100°C for 5 min for denaturation and electrophoresis separation was performed at 90V. Following the electrophoresis, the protein was transferred onto a PDVF membrane using constant current of 300 mA. The PDVF film blocking was carried out at room temperature for 2 h using TBST blocking solution containing 5% skimmed milk. After the blocking agent was removed, primary antibodies were diluted and added for culture overnight on a 4°C shaker. After three cycles of TBST rinsing, each time for 8 min, secondary antibodies (AS014, ABclonal) were provided, incubated at room temperature for 1.5 h, and followed by a cycle of TBST washing. Ultimately, ECL chemiluminescence detection kit with a Tanon-4500 chemiluminescence system are used for development. The primary antibodies included AKT (A11016, ABclonal); p-AKT (AP0637, ABclonal); IKK (A2062, ABclonal); p-IKK (AP0505, ABclonal); NF-κB (10745-1-AP, ABclonal); p–NF–κB (3033T, ABclonal); and β-Actin (AC026, ABclonal).