Glass culture slides

Cells were seeded on glass culture slides (BD Biosciences) and fixed with 4% cold methanol at − 20 °C for 10 min. Subsequently, cells were blocked with 10% goat serum for 1 h and incubated with primary antibodies against YAP (Cell Signaling Technology, Cat. 14,074), TAZ (Abcam, Cat. ab224239) or Phalloidin-iFluor 488 Reagent - CytoPainter (Abcam, Cat. ab176753) at room temperature for 2 h and then incubated with secondary antibody FITC for 1 h at room temperature. After counterstained with DAPI (Invitrogen), the slide was observed under a confocal microscope (Zeiss).

Cells were grown on glass culture slides (BD Biosciences, San Jose, USA) and fixed with 4% cold methanol at -20°C for 10 min. Subsequently, cells were blocked with 10% goat serum for 1 h and incubated with primary antibodies against β-catenin (ab32572, Abcam, Cambridge, UK, 1:250) or RAI2 (PA5-62305 Invitrogen, CA, USA, 1:500) at 4°C for 1 h and then incubated with fluorescent labeled secondary antibodies for 1h at room temperature. After being counterstained with DAPI (Invitrogen, CA, USA), the slide was observed under a confocal microscope (Zeiss).

Cells were seeded at 0.7–2.5 × 10⁴ cells/1.7 cm² well of glass culture slides (BD Falcon, Bedford, USA). Cells were transfected as previously described. The cells were fixed in 4% parafomaldehyde (Sigma-Aldrich, Steinheim, Germany), in PBS for 20 min. The cells were then permeabilized in 0.5% Triton X-100 in PBS for 5 min, washed in PBS and blocked in blocking solution (1% BSA, 10% donkey serum [both from Sigma, St. Louis, USA] in PBS) for 1 h at RT. Transfected proteins and cell organelles were stained with appropriate antibodies or counter stains according to manufacturer’s protocol. Antibodies and co-stains were as follows: mouse anti-EGFR (GR01L, 1:1000, Merck-Calbiochem, Darmstadt, Germany), mouse anti-transferrin receptor (1:100, Invitrogen, Camarillo, USA), donkey anti-mouse Alexa Fluor 488 (1:200, Invitrogen-Molecular Probes, Eugene, USA). The slides were sealed with a coverslip and Prolong Gold antifade mounting media with DAPI (Life Technologies-Molecular Probes, Eugene, USA). Slides were viewed on a Leica SP8 Confocal Microscope. Fluorescent images were captured with a 63x lens zoomed 1-4x with a 1024 × 1024 frame and 400 Hz scanning speed. Images were analyzed using Leica LAS X software. The images presented in the same figures were captured using standardized setting and exposure times.

Cells were seeded on glass culture slides (BD Falcon, Franklin Lakes, New Jersey, USA) and cultured for 24 h. Subsequently, they were fixed with 4% paraformaldehyde, permeabilized with 0.25% Triton X‐100, and blocked with 1% BSA. For cytoskeleton detection, F‐actin was detected with phalloidin‐Alexa Fluor 488 (Thermo Fisher Scientific, Carlsbad, California, USA). For the detection of protein localization, immunostaining was performed with FLAG (1:100), ARHGAP35 (1:100), and TFII‐I (1:100) primary antibodies, and subsequently the appropriate secondary antibodies. The nuclei were stained with 4′,6‐diamidino‐2‐phenylindole (DAPI) (Thermo Fisher Scientific). Images were acquired using an Olympus FluoView FV1000 confocal microscope.