Anti β catenin mab

The following primary monoclonal antibodies (mAbs) and polyclonal antibodies (pAbs) were used to detect junctional and signaling proteins by immunofluorescence labeling and immunoblotting: anti-occludin, anti–ZO-1, and anti–E-cadherin mAb (Thermo-Fisher Scientific, Waltham, MA); anti–β-catenin mAb (BD Bioscience, San Jose, CA), Anti-claudin 1 and 4 pAbs (Abcam, Cambridge, UK), anti-cortactin mAb (p80/85 clone 4F11, EMD Millipore, Billerica, MA), anti-cortactin pAb (H222), anti-phospho-CREB (Ser133; 87G3) rabbit mAb, and anti-CREB (48H2) rabbit mAb (Cell Signaling Technologies, Danvers, MA), anti-GAPDH mAb (6C5, Abcam, Cambridge, MA). Fluorescently labeled phalloidin 488 (Thermo-Fisher Scientific) was used to visualize actin filaments. Anti-rabbit and anti-mouse secondary antibodies conjugated to Alexa-488 or Alexa-568 dyes were obtained from Thermo-Fisher Scientific. Mouse and rabbit secondary HRP-conjugated antibodies were purchased from GE Healthcare (Pittsburgh, PA).

Anoikis-induced cells were pelleted, washed, and fixed in 4% paraformaldehyde. Fixation was quenched using 100 mM glycine pellets, which were processed for cryo-sectioning using OCT media and a cryostat. ∼8–10-μm thick sections on slides were used for immunocytochemistry and PLA studies. Briefly, sections were permeabilized with 2% Triton X-100, blocked in 10% donkey serum, and then incubated overnight in primary antibodies (anti-β-catenin mAb [Cat #610154; BD Biosciences], anti-YAP1 mAb [Cat #52771; Abcam], and anti- MUC13 [Cat#NBP2-25466; Novus]). After washing, samples were incubated with Cy3 anti-rabbit and Alexa 488 anti-mouse secondary antibodies. Slides were mounted with Vector Shield with DAPI (Cat#H-2000; VECTASHIELD), and images were captured at 400x magnification using a confocal microscope (Nikon Corporation). Images were analyzed using free Zen software provided by Zeiss and Image J software.