Egfr y1068

In total, 1.2×10⁶ cells/10-cm dish were plated 24 h before treatment. After hyperthermia and cisplatin treatment for 2 h, the spent media was replaced with 10 mL fresh media, and the cells were cultured for 24 h at 37°C. The floating apoptotic cells were then collected through centrifugation, and the attached cells were collected through trypsinization. The combined cell pellets were homogenized with lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 10% glycerol) on ice for 45 min. The following antibodies were used for WB: EGFR (4267, Cell Signaling Technology); EGFR-Y1068 (3777, Cell Signaling Technology); AKT (9272, Cell Signaling Technology); AKT-S473 (4060, Cell Signaling Technology); P53 (2527, Cell Signaling Technology); P53-S15 (9286, Cell Signaling Technology);; PARP (9542, Cell Signaling Technology); Bim-1 (2933, Cell Signaling Technology); β-actin (A5316, Sigma-Aldrich); and Bax (2772, Cell Signaling Technology).

ALK small-molecule inhibitors PF-02341066 (Crizotinib) and NVP-TAE684 were purchased from Selleckchem (Selleck Chemicals, Houston, TX, USA), whereas anti-ALK monoclonal antibodies (mAb46 and 30) were a generous gift of Dr. Marc Vigny [39 (link)]. The primary antibodies for Western blot analysis were from Cell Signalling (ALK^Y1604; AKT^S473; ERK^T202/Y204; c-Met^Y1234/1235; EGFR^Y1068; IGF1-Rβ^Y1135/1136; IGF1-Rβ) (Cell Signaling Technology, Inc., Danvers, MA, USA); SIGMA (γ-tubulin) (SIGMA Aldrivh Co., USA); Invitrogen (ALK) (Invitrogen, Life Technologies Co); or Santa Cruz (AKT; ERK) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Horseradish peroxidase (HRP)-conjugated sheep anti-mouse or donkey anti-rabbit antibodies, were purchased from GE Healthcare (GE Healthcare Life Sciences, Uppsala, Sweden), whereas recombinant human HGF, EGF, and IGF-1 growth factors were purchased from Peprotech (Peprotech, NJ, USA). Pervanadate stock solution (100mM) was prepared as indicated by Huyer et al. [40 (link)]; while MTT salt (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was available from SIGMA (SIGMA Aldrich Co., USA).