Beta catenin

The human prostate cancer cell lines, PC3, DU145, and LNCaP Clone FGC, were obtained from the National Collection of Authenticated Cell Cultures (Shanghai, China) and were STR certified. PCR was used to detect Mycoplasma in the culture medium, and the passage time of the cells was not more than 6 months. The cell lines were cultured in RPMI 1640 (HyClone, USA) supplemented with 10% fetal bovine serum (FBS, Gibco) and grown at 37 °C, 5% CO₂. The antibodies included TACR2 (Proteintech, 25270-1-AP), beta-Catenin (Proteintech, 51067-2-AP), GAPDH (Cell Signaling Technology, 5174S), Cyclin D1 (Cell Signaling Technology, 55506S), Lamin B1 (Cell Signaling Technology, 13435S).

The total proteins of SW480 and NCM460 cells were prepared using RIPA buffer containing protease and phosphatase inhibitors. A BCA protein assay kit was used to measure protein concentrations. 20 µg proteins were loaded per lane, separated by electrophoresis, and then transferred to polyvinylidene fluoride (PVDF) membranes (C3117, Millipore). The membrane was blocked and then incubated for 1 h with β-actin (1:100000; ABclonal, AC026), PPAR gamma (1:5000; Proteintech, 16643-1-AP), MMP9 (1:1000; Proteintech, 10375-2-AP), TNF alpha (1:3000; Proteintech, 60291-1-IG), TGF beta1 (1:2000; Proteintech, 21898-1-AP), COX2 (1: 500; Proteintech, 27308-1-AP), HIF1A (1:2500; Proteintech, 20960-1-AP) and Beta-catenin (1:10000; Proteintech, 51067-2-AP). Immunoblot analysis was performed with horseradish peroxidase (HRP)-conjugated anti-mouse antibodies or anti-rabbit antibodies (1:5000; ZSGB-BIO, ZB-5301, and ZB-5305) and developed with the ECL kit (Beyotime Biotechnology, P0018FM). The level of β-actin was used as a loading control, and the ratios of the gray value of the target protein bands to the gray value of the corresponding internal control bands were defined as the expression level of the target protein.

Western blot was performed as previously described (27 (link)). Samples of equivalent total protein (40 μg) were loaded. Primary antibody against REST, p-Raf-1, p-beta-catenin, p-MEK-2, p-ERK-1/2 (Abcam, Cambridge, UK, 1:500), E-cadherin, N-cadherin, Vimentin, MMP-9, ZO-1, MEK-2, ERK1/2, Raf-1, beta-catenin, beta-Actin, and Histone H3 (ProteinTech Group, Rosemont, USA, 1:500) were used. Suppression of MAPK signaling pathway with U0126 was performed as previously described (28 (link)). In brief, cells incubated in t25 culture flask with 60~70% confluency were treated with 10 μM U0126 for 36 h before further analysis.