Anti e cadherin clone 36

The following primary mouse monoclonal antibodies were used: anti-E-cadherin, clone 36; anti-β-catenin, clone 14; anti-paxillin, clone 349 (BD Transduction Labs, Franklin Lakes, NJ, USA); anti-zyxin, clone 164D4 (Synaptic Systems, Göttingen, Germany); anti-β-actin, clone 4C2 (Merck, Darmstadt, Germany); anti-vinculin, clone hVin1 (Sigma, Merck, Darmstadt, Germany); anti-α-actinin-1, clone BM-75.2 (Sigma, Merck); anti-α-catenin, clone 15D9 (Enzo Life Sciences, Farmingdale, NY, USA); rabbit polyclonal anti-p34-Arc/ARPC2 (Upstate, Merck, Darmstadt, Germany); anti-pY654-β-catenin (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). The secondary goat anti-mouse IgG, IgG1, IgG2a, IgG2b or anti-rabbit IgG antibodies conjugated with AlexaFluor488, 594 or 647 (Jackson Immunoresearch, West Grove, PA, USA) were used. AlexaFluor488-conjugated phalloidin (Molecular Probes, Eugene, OR, USA) or TRITC-conjugated phalloidin (Fluka Chemie, Buchs, Switzerland) was added to the secondary antibodies. For Western blotting, goat anti-mouse and anti-rabbit IgG antibodies conjugated to horse radish peroxidase (Jackson Immunoresearch) were used. Other reagents were obtained from Sigma, Merck.

Protein lysates and conditioned media (CM) samples were prepared as previously described (Kamata et al., 2015 (link), 2020 (link)). Membrane protein purification was performed using a ProteoExtract^® Native Membrane Protein Extraction Kit (Merck) according to the manufacturer's instructions. Immunoblotting and enzyme-linked immunosorbent assay (ELISA) were performed as previously described (Kamata et al., 2015 (link), 2020 (link)). Primary antibodies used for immunoblotting were as follows: anti-CCL6 (ab83134, Abcam; 1:2000), anti-P-AKT (clone D9E, Cell Signaling Technology; 1:2000), anti-pan-AKT (clone C67E7, Cell Signaling Technology; 1:5000), anti-RAC1 (ARC03, Cytoskeleton, Inc.; 1:2000), anti-RHOA (ARH04, Cytoskeleton, Inc.; 1:2000), anti-CDC42 (ACD03, Cytoskeleton, Inc.; 1:2000), anti-pan-RAS (clone EP1125Y, Merck; 1:2000), anti-E-cadherin (clone 36, BD Biosciences; 1:1000), anti-GAPDH (clone GA1R, Thermo Fisher Scientific; 1:4000) and anti-MAC2 (CL8942AP, Cedarlane; 1:2000) antibodies. Antibody validation profiles were provided by respective companies upon purchase.

The following mouse monoclonal antibodies were used as primary antibodies: anti-phospho-ERK1/2 (Thr202/Tyr204) (clone E10, #9106; Cell Signaling Technologies, MA, USA), anti-β-actin (clone AC-15, #A5441; Sigma-Aldrich Corporation, MO, USA), anti-desmoplakin (clone 11-5F; gift from David Garrod, University of Manchester, UK), anti-keratin 14 (clone LL001; Purkis et al., 1990 (link)), anti-keratin 10 (clone DE-K10, #M7002; Agilent Technologies, CA, USA), anti-E-cadherin (clone 36, #610182; BD Biosciences, CA, USA) and anti-Ki-67 (clone MM1, #NCL-L-Ki67-MM1; Leica Biosystems, IL, USA). The following rabbit monoclonal or polyclonal antibodies were also used as primary antibodies: anti-phospho-AKT (Ser473) (clone D9E, #4060), anti-AKT (clone C67E7, #4691), anti-phospho-ERK1/2 (Thr202/Tyr204) (clone D13.14.4E, #4370), anti-ERK1/2 (clone 137F5, #4695) and anti-YAP (clone D8H1X, #14074) were purchased from Cell Signaling Technologies (MA, USA). Secondary antibodies used were as follows: goat anti-rabbit IgG (H+L) Alexa Fluor 594 (#A-11037) and goat anti-mouse IgG (H+L) Alexa Fluor 568 (#A-11031) were purchased from Thermo Fisher Scientific (MA, USA), anti-mouse IgG (H+L), HRP conjugate (#W4021) and anti-rabbit IgG (H+L), HRP conjugate (#W4011) were purchased from Promega Corporation (WI, USA).