Pegfr

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].

Cells were lysed in RIPA Lysis buffer (Santa Cruz Biotech) or 1X Blue loading buffer (Cell Signaling). Protein lysates (25-100μg) were resolved on SDS-PAGE and transferred onto nitrocellulose membranes using a standard western blot protocol. Membranes were probed with primary antibodies against E-cadherin, N-cadherin, fibronectin, vimentin, ZO-1, EGFR, p-EGFR, (BD Biosciences), CXCR1 (Thermo Scientific), GAPDH (Santa Cruz), AXL, p-AXL (R&D Systems), p38, p-p38 (Cell Signaling), c-MET (Invitrogen) and brachyury (MAb 54-1) [50 (link)], at 4°C overnight. Membranes were incubated with an appropriate secondary antibody conjugated with IRDye and detected by the Odyssey infrared detection method (Li-COR Biotechnology). For detection of phospho-MAPK in tumor lysates of HCC827 and A549 tumor cell pairs, a Proteome Profiler Human Phospho-MAPK Array Kit (R&D Systems) was used, following the manufacturer's recommendations. Signal was detected and quantified by the Odyssey infrared detection method (Li-COR Biotechnology).