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).
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