Erk1 erk2

The lysates were loaded on SDS-PAGE gels, followed by blotting onto polyvinylidene difluoride membranes (BioRad Laboratories, Inc., CA, USA). The following antibodies were obtained from Cell Signaling Technology (MA, USA): GAPDH (14C10) (cat # 2118, 1:1000 dilution), Lamin B1 (D4Q4Z) (cat# 12586, 1:1000 dilution) (Cell Signaling Technology), IGF1 Receptor β (D23H3) XP® (cat # 9750, 1:1000 dilution), Phospho-IGF1 Receptor β (Tyr1131)/Insulin Receptor β (Tyr1146) (cat # 3021, 1:1000 dilution), AKT (cat # 9272, 1:1000 dilution), Phospho-AKT (Ser473) (cat # 9271, 1:1000 dilution). The antibodies ERK1/ERK2 (cat # MAB1576, 0.5 µg/mL dilution) and Phospho-ERK1 (T202/Y204)/ERK2 (T185/Y187) (cat # MAB1018, 0.5 µg/mL dilution) were obtained from R&D Systems Inc. (MN, USA). The anti-BCOR antibody (cat# ab88112, 0.5 µg/mL dilution) was purchased from Abcam (Cambridge, UK). The HRP-linked secondary antibodies were obtained from Cell Signaling Technology (MA, USA) (anti-mouse IgG (cat# 7076)) and Sera Care (MA, USA) (anti-rabbit IgG (cat# 074-1516)). Detection was done by SuperSignal™ West Dura Extended Duration Substrate (Thermo Fisher Scientific, MA, USA), and imaging was performed on Fusion Pulse TS (Vilber Lourmat, Eberhardzell, Germany).

Sodium dodesyl sulfate (SDS) polyacrylamide gels (Bio-Rad, Hercules, CA) were loaded with 40 μg total protein per lane; following electrophoresis, the proteins were transferred onto polyvinylidene difluoride membranes (Bio-Rad), which were incubated with Blocking One (Nacalai Tesque, Kyoto, Japan) for 1 h at room temperature, followed by overnight incubation at 4°C with anti-ALK (C26G7), anti-phospho-ALK (Tyr1604), anti-phospho-EGFR (Tyr1068), anti-STAT3(79D7), anti-phospho-STAT3 (Y705), anti-AKT, anti-phospho-AKT (Ser473), anti-ErbB4 (111B2), anti-phospho-ErbB4 (Tyr1284), anti-MET (25H2), anti-phospho-MET (Y1234/Y1235) (3D7), or anti-β-actin (13E5) antibodies (1:1,000 dilution each; Cell Signaling Technology, Danvers, MA), or with anti-human EGFR (1 μg/mL), anti-human/mouse/rat extracellular signal-regulated kinase (Erk)1/Erk2 (0.2 μg/mL), or anti-phospho-Erk1/Erk2 (T202/Y204) (0.1 μg/mL) antibodies (R&D Systems). After washing 3 times, the membranes were incubated for 1 h at room temperature with secondary antibodies (horseradish peroxidase-conjugated species-specific antibodies).
Immunoreactive bands were visualized with SuperSignal West Dura Extended Duration Substrate Enhanced Chemiluminescent Substrate (Pierce, Osaka, Japan). Each experiment was independently carried out at least 3 times.

Lysates were prepared using Cell Lysis Buffer (Cell Signaling, Danvers, MA). Immunoblotting was performed as previously described 13. Antibodies used in this study were as follows: anti‐EGFR, anti‐phospho‐EGFR (Tyr1068), anti‐MET, anti‐phospho MET (Tyr1234/1235), anti‐TRK, anti‐phospho TRK (Tyr490), anti‐AKT, anti‐phospho‐AKT (Ser473), and anti‐β‐actin (13E5) antibodies (each used at a 1:1000 dilution; Cell Signaling Technology, Danvers, MA). Additional antibodies were also used, including the anti‐human/mouse/rat extracellular signal‐regulated kinase ERK1/ERK2 (0.2 μg/mL) and the anti‐phospho‐ERK1/ERK2 (T202/Y204) (0.1 μg/mL) from R&D Systems. The immunoreactive bands were visualized using the SuperSignal West Dura Extended Duration Substrate, an enhanced chemiluminescent substrate (Pierce Biotechnology, Rockford, IL). Each experiment was performed independently at least three times.