Mouse anti ns1

The cells were lysed with an appropriate volume of protein lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA) containing phosphatase and protease inhibitors (Thermo Fisher Scientific, Waltham, MA, USA). The protein concentration was then determined using the Bio‐Rad Protein Assay Kit (Bio‐Rad, CA, USA). Ten micrograms of each protein sample were electrophoresed by SDS‐PAGE and transferred onto a nitrocellulose membrane. Western blot analysis was performed using the following primary antibodies and dilutions: mouse anti‐NP (ATCC, HB‐65, 1:50), mouse anti‐NS1 (Santa Cruz biotechnology, Santa Cruz, CA, USA, 1:1000), mouse anti‐β‐actin (Thermo Fisher Scientific, 1:3000), rabbit anti‐β‐catenin (Cell Signaling, Beverly, MA, USA, 1:1000), rabbit anti‐LEF1 (Cell Signaling, 1:1000), and rabbit anti‐FZD4 (Cell Signaling, 1:1000). The membranes were incubated with primary antibodies overnight at 4°C, followed by incubation with horseradish peroxidase (HRP)‐conjugated goat anti‐mouse IgG (Jackson ImmunoResearch, PA, USA, 1:2000) or goat anti‐rabbit IgG (Jackson ImmunoResearch, 1:2000) at room temperature for 1 hr. Protein bands were visualised using the enhanced chemiluminescence kit (Thermo Fisher Scientific, Rockford, IL, USA).