Phospho specific antibodies against

Phospho-specific antibodies against ERK (Thr-202 and Tyr-204), Akt (Ser-473), p90RSK (Ser-380), EGFR (Thr-669), ErbB2 (Tyr-1196 and Tyr-1248) and ErbB3 (Tyr-1289), and total EGFR antibody were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against EGFR, ErbB2, ErbB3, Akt1 and Actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Recombinant human epidermal growth factor (EGF), heregulin (HRG), and fibroblast growth factor-2 (FGF-2) were purchased from R&D Systems (Minneapolis, MN, USA). SB203580, SP600125, U0126, and LY294002 were obtained from Merck Biosciences (Darmstadt, Germany). Lapatinib and trametinib were obtained from AdooQ BioScience (Irvine, CA, USA) and Cayman Chemical (Ann Arbor, MI, USA), respectively. SCH772984 was purchased from Chemietek (Indianapolis, IN, USA). TPA and Phos-tag ligand were purchased from WAKO Pure Chemical Industries (Osaka, Japan). All chemical agents were dissolved in DMSO, and the final concentration of DMSO was less than 0.1%.

Clonetics EGM-2 MV BulletKit and fetal bovine serum (FBS) were purchased from Takara Bio Inc. (Shiga, Japan). An EZ-Cytox Enhanced Cell Viability Assay Kit was purchased from ITSBIO (Seoul, Korea), and deoxyshikonin was purchased from Sigma-Aldrich (St. Louis, MO, USA). Mayer’s hematoxylin was obtained from Muto Pure Chemicals (Tokyo, Japan). Phospho-specific antibodies against ERK (Thr-202/Tyr-204), p38 (Thr-180/Tyr-182), Akt (Ser-473) and antibodies against Akt, VEGFR2 (60A8), and VEGFR2 (Y1059) were purchased from Cell Signaling Technology (Denvers, MA, USA). Antibodies against ERK1 (C-16), p38 (C-20), VEGF, and β-actin (I-19) were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA, USA). Chemical inhibitors SB 203580 and U0126 were obtained from Calbiochem (Darmstadt, Germany). All chemical inhibitors were dissolved in dimethyl sulfoxide (DMSO) prior to use, and the final concentration was kept <0.1% for each experiment.