α e catenin

Affinity purification using anti-FLAG agarose (Millipore Sigma, St. Louis, MO, USA), glutathione-agarose (Millipore Sigma), and TALON resin (Clontech, Mountain View, CA, USA) was done as described [16 (link)–19 (link)]. Immunoblot analysis was performed using antibodies purchased from Santa Cruz Biotechnology [(Myc tag (9E10), sc-40; actin, sc-1616-R; dynein heavy chain, sc-9115; ß-catenin, sc-7199; γ-catenin, sc-7900; Src, sc-18; desmoplakin, sc-33555; c-Myc, sc-764; Enigma, sc-98370; Galectin 1, sc-28248; EGFR, sc-03; phosphotyrosine (PY99), sc-7020], Cell Signaling Technology (Beverly, MA, USA) [EGFR, #4267; c-Met, #3127; HER2, #2165; PKCδ, #2058; phospho-PKCδ[Y311], # 2055; CDCP1, #4115 and #13794; P-CDCP1[Y707], #13111; P-CDCP1[Y734], #9050; P-CDCP1[Y743], #13093; P-CDCP1[Y806], #13024; β1-integrin, #4706; MMP14, #13130; P-EGFR[Y845], #6963; P-EGFR[Y992], #2235; P-Src[Y416], #6943; p62, #5114; α-E-catenin, #3236], BD Transduction Laboratories (San Jose, CA, USA) [E-cadherin, 610182; plasminogen activator inhibtor-1 (PAI-1), 612024], BD Biosciences (San Jose, CA, USA) [p120 catenin, 610133], Neomarkers (Freemont, CA, USA) [cyclin D1, MS-210], EMD Millipore (Temecula, CA, USA) [anti-phosphotyrosine (4G10), 05-321], Invitrogen [desmoglein 2, 32-6100], Qiagen (Valencia, CA, USA) [anti-His₅, 34660], and Millipore Sigma [anti-FLAG (M2), F3165].

For immunofluorescence, paraffin sections were rehydrated, and antigen retrieval was performed with citrate buffer containing 2 mM EDTA, 0.05% Tween 20 in a pressure cooker. Antibodies and dilutions used were as follows: αE-catenin (mouse, 1:500; BD Transduction Laboratories), β-catenin (rabbit, 1:1,000; Abcam), BrdU (rat, 1:500; Invitrogen), Ki67 (rat, 1:100; DAKO), GFP (rabbit, 1:1,000; Abcam), p21 (mouse, 1:500; BD Transduction Laboratories), YAP (rabbit, 1:100; Cell Signalling), and pYAP (rabbit, 1:100; Cell Signalling), E-cadherin (rat, 1:1,000; Zymed), Scribble (goat, 1:100; Cell signalling), PAR3 (rabbit, 1:500; Millipore). Secondary antibodies were coupled to Alexa 488, Alexa 555 (Invitrogen), or Biotin (Vector). TSA kit (PerkinElmer) was used for signal amplification for p21, YAP and pYAP detection. Nuclear counterstaining was performed with DAPI (Invitrogen), and all images were acquired using a Leica-TCS SP5 confocal microscope.