6 well culture dishes

EBs in differentiation medium were collected from 9 to 17 days in vitro (DIV) in a 15 ml tube. Then EBs were centrifuged at 300 g for 5 min, differentiation medium was removed and EBs were re-suspended in NPC medium containing: DMEM/F12 (Gibco), N2 supplement (1%, Invitrogen), B27-RA supplement (1:50, Invitrogen), laminin (1 µg/ml, Sigma-Aldrich), mouse bFGF (20 ng/ml, Invitrogen), penicillin/streptomycin (1%, Sigma-Aldrich). EBs re-suspended in NPC medium from one 96 well plate were split into two 10 cm² wells of 6-well culture dishes (Greiner Bio-One), which were coated with laminin (50 ug/ml, Sigma-Aldrich) for at least 30 min at 37 °C. NPCs were seeded immediately after removing excess of laminin. Adherent cultures were refreshed every other day with NPC medium and passaged (1:4) every 4 days for up to 6 passages. NPCs could be preserved at every passage after washing with PBS, detaching with 0.25% trypsin-EDTA (Sigma-Aldrich) for 5 min at 37 °C, resuspension in DMEM/F12 (Gibco) with 10% FBS (Lonza), centrifugation at 300 g for 5 min, followed by resuspension of NPCs in FBS containing 10% DMSO (Sigma-Aldrich) in cryotubes.

pIRESneo-MET or pIRESneo-MET^Δ7–8 were transfected into HEK-293T or TOV-112D cells in 6-well culture dishes (Greiner Bio-One, Krëmsmunster, Austria) using Fugene HD transfection reagent (Promega, Fitchburg, WI, USA) according to the manufacturers’ instructions. After 48 h, cell monolayers were washed with PBS and cell extracts prepared in RIPA buffer containing protease and phosphatase inhibitors (Cell Signaling Technology, CST, Danvers, MA, USA).
In separate experiments, pHLsec-MET^Δ7–8ED-BAPHIS or pHLsec-MET^ED-BAPHIS were cotransfected in a 1:1 ratio with the biotin-ligase expression construct pDISPLAY-BirA-ER (Addgene) in HEK293T cells and cells were cultured in the presence of 10 μM biotin (Sigma-Aldrich, St Louis, MO, USA). After 48 h, cytosolic extracts were made in RIPA buffer, and medium (1 ml) was mixed with 100 μl Ni–NTA Sepharose slurry (IBA, Goettingen, Germany). After a 1 h incubation at 4 °C, Ni-beads were washed with buffer (500 mM NaCl, 50 mM phosphate buffer, pH 7.4) and loaded onto a poly-prep column (Bio-Rad, Hercules, CA, USA). After washing off specifically bound proteins with 2 ml 10 mM imidazole, His-tagged an biotinylated MET extracellular domains were eluted with 0.5 ml 0.5 M imidazole, and dialysed o/n at 4 °C to 50 mM TRIS/150 mM NaCl pH 7.5.