Polyclonal goat anti rabbit immunoglobulins

For each PDX model, four tumors were randomly selected and tumor lysates were prepared from snap frozen tumor fragments as described previously [38 (link)]. The lysates were then loaded on NuPAGE™ 4%–12% Bis-Tris Gels (ThermoFisher Scientific) and electrophoresed using NuPage™ MOPS SDS Running Buffer (ThermoFisher Scientific) at 150 V for 2 h. Four different tumor lysates were used per treatment group. Samples were then blotted on polyvinylidene fluoride membranes (Bio-Rad) in a semi-dry transfer at 25 V for 30 min, using a transfer buffer with 20% methanol, 25 mM tromethamine and 190 mM glycine. The following primary antibodies were used: KIT (A450229-2, Agilent), phospho-KIT Tyr719, phospho-KIT Tyr703, AKT, phospho-AKT Ser743, p44/42 mitogen-activated protein kinase (MAPK), phospho-p44/42 MAPK Thr202/Tyr204, eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1), phospho-4E-BP1 Ser65, ribosomal protein S6, phospho-S6 Ser240/244 and α-tubulin (catalogue numbers: 3391, 3073, 7292, 7291, 9102, 4370, 9644, 9456, 2217, 5364 and 2144, respectively; all from Cell Signaling Technology). Horseradish peroxidase conjugated secondary polyclonal goat antirabbit immunoglobulins (P044801, Agilent) were added and specific bands were visualized using Western Lightning™ ECL Pro kit (Perkin Elmer). Chemiluminescence was captured using the FUJI-LAS mini 3000 system (Fujifilm).