Fluorescent dye conjugated secondary antibody

Cells were grown to 80% confluence on glass coverslips. Then, the cell layers were washed with PBS and fixed with 4% paraformaldehyde (PFA). Cells were incubated with ice-cold 100% methanol for 10 min at −20°C. After rinsing with PBS for 5 min, the cells were incubated with blocking buffer (1 × PBS/5% normal serum/0.3% Triton™ X-100) for 60 min. The anti-β-catenin antibody (1:100 dilution, Cell Signaling Technology) was diluted in antibody dilution buffer (1 × PBS/1% albumin from bovine serum albumin (BSA)/0.3% Triton™ X-100). Cells were incubated with diluted primary antibodies overnight at 4°C. After thorough washing, cells were incubated with fluorescent dye-conjugated secondary antibodies (1:1,000 dilution, Cell Signaling Technology) for 1 h at room temperature. Finally, cells were washed with PBS and stained with DAPI (Thermo Fisher Scientific) for 10 min at room temperature in the dark. Slides were covered with mounting medium, and images were captured under a Leica TCS SP8 fluorescence confocal microscope (Wetzlar, Germany).

Actin‐stain™ 670 phalloidin (Cytoskeleton, Denver, CO, USA) was used for cytoskeletal F‐actin staining. Approximately 2 × 104 cells in 1 mL of RPMI‐1640 medium with 10% FBS were seeded on a 20‐mm confocal dish. On the next day, the cells were gently washed once with PBS at 37 °C and fixed in a fixative solution for 10 min. After washing, permeabilizing and blocking, 200 µL of 200 nm Actin‐stain™ 670 phalloidin was added to the cells and they were incubated in the dark at room temperature for 30 min. For SIK2 and p‐MYL2 staining, the primary antibody was incubated overnight at 4 °C, followed by the incubation of the fluorescent dye‐conjugated secondary antibody (Cell Signaling Technology, Danvers, MA, USA) for 1 h. Subsequently, DNA was counterstained for 30 s with 200 µL of 100 nm DAPI in PBS. Fluorescent images were visualized using a laser confocal microscope (LSM880; Carl Zeiss, Oberkochen, Germany).