β catenin

BCA kit was used to determine the protein concentration. Proteins were then electrophoresed on sodium dodecyl sulfate-polyacrylamide gels (10%) and transferred to a polyvinylidene fluoride membrane. Primary antibodies against HMGB3 (1:1000; Proteintech Group), CD63 (1:5000; Proteintech Group), tumor susceptibility 101 (TSG101; 1:2000; Proteintech Group), β-catenin (1:2000; Proteintech Group), c-Myc (1:2000; Proteintech Group), non-phospho (active) β-catenin (1:1000; Abcam), and β-actin (1:1000; Proteintech Group) were used to incubate the membranes overnight at 4°C. Then, the membranes were incubated with the appropriate secondary antibodies for 1 h. An ECL kit was used for protein detection.

Total protein was extracted by RIPA (Solarbio, Beijing), and the concentration was measured by bicinchoninic acid (Solarbio). The nuclear and cytoplasmic fractions were isolated by a Nuclear Extraction Kit (Solarbio, Beijing, China) according to the manufacturer‘s instruction. 80 μg/well proteins were loaded onto 10% SDS-PAGE for each sample, and proteins were transferred onto equilibrated polyvinylidene difluoride membranes (Millipore Corporation, Billerica, MA, USA) by electroblotting. Membranes were blocked with TBST containing 5% milk and incubated overnight at 4°C with primary antibodies against HO-1 (Bioworld Tech, LP, USA), Wnt1 (Abcam, Cambridge, MA, USA), Wnt5a (Novus Biologicals, Littleton, USA), β-catenin, GSK-3β, phospho-GSK-3β (Ser9) (ProteinTech Group, Chicago, USA), and NFAT5 (Santa Cruz, CA, USA). After incubation with the secondary antibody (ProteinTech), proteins expression was corrected by the amount of β-actin (ProteinTech) in the same sample. Lamin A (ProteinTech) and tubulin (ProteinTech) were used as the marker for nuclear and cytosolic proteins, respectively. The bands were quantified by scanning densitometry using Quantity One 4.6.3 software (Bio Rad).

The cells were fixed with 4% paraformaldehyde for 30 min and then kept stable in 0.2% Triton X-100 for 30 min to rupture cell membranes. Subsequently, the cells were blocked with 2% bovine serum albumin for 30 min. The cells were then incubated overnight at 4 °C with antibodies against BAP31 antibody (1:200, Abcam, Cambridge, UK) and β-catenin (1:200, Proteintech, Wuhan, China). After washing, the cells were further incubated with the appropriate fluorescence-conjugated secondary antibody (CST, Danvers, MA, USA) for 1 h at room temperature and stained with DAPI (Beyotime). They were then examined by fluorescence microscopy (Leica).