Phospho rtk array

Cell samples were lysed in RIPA buffer (50 mM Tris pH7.4, 150 mM NaCl, 1% NP40, 0.1% SDS and 0.5% Sodium deoxycholate) supplemented with protease inhibitor (Roche, #11836153001) and phosphatase inhibitor cocktails 2 and 3 (Sigma-Aldrich, #P57261 and #P0044). Protein concentrations were determined by Pierce BCA protein assay kit (Thermo Scientific, #23225). Proteins were separated by SDS-PAGE in Laemmli buffer (0.25 M Tris, 1.92 M glycine, 1% SDS), transferred to PVDF membranes (Carl Roth, pore size 0.45 μM) in transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol) and subsequently incubated overnight at 4 °C with indicated antibodies in 5% BSA in PBST. ECL (Perkin Elmer, NEL104001EA) was used to detect antibodies in a Vilber Fusion FX. Western blotting experiments were performed three times and representative results are shown.
The human Phospho-RTK Array was purchased from R&D Systems (ARY001B) and was used according to the manufacturer’s protocols. Cells were seeded in 10 cm dishes, allowed to adhere overnight and subsequently treated as indicated.

The phospho-RTK array (R&D Systems) specifically screens for membrane associated receptors. Antibodies against 42 different RTKs were pre-spotted in duplicate on nitrocellulose membranes. Cell lysates from SCC1 ShLacZ and ShTrop2 cells were incubated with the membrane according to the manufacturer's instructions. Thereafter, a pan anti-phosphotyrosine antibody was used to detect the phosphorylated tyrosine on activated RTKs. The blots were also developed using the ECL method.