Sult2b1

Samples were lysed in RIPA buffer (Beyotime, Shanghai, China) containing protease inhibitor cocktail, phosphatase inhibitors and phenylmethylsulfonyl fluoride (PMSF). Equal amounts of protein (30–50 μg) were loaded and separated on a 10% SDS-polyacrylamide gel, electrotransferred onto PVDF membranes (Millipore, Billerica, MA, USA), and blocked in 5% bovine serum albumin in Tris-buffered saline containing Tween 20 for 1 h at room temperature. The membranes were probed with primary antibodies against p-AKT (S473), AKT, CCND1, CCNA2, CLND1, CDH1 (Cell Signaling Technology, MA, USA), HSP90 (Proteintech, IL, USA) and SULT2B1 (R&D systems, MN, USA) overnight at 4 °C. After three 10 min washes with Tris-buffered saline with Tween-20 (TBST), the membrane was incubated at room temperature for 1 h with an appropriate secondary antibody conjugated to horseradish peroxidase. HSP90 was used as the loading control. The protein signals were visualized with a Pierce ECL Western Blotting kit (Life Technologies, Thermo Fisher Scientific, USA).