Anti etv5

IMR-32 and SK-N-BE(2)C cell lines were obtained from the American Type Culture Collection (ATCC). CLB cell lines were derived by V. Combaret (Lyon, France). Cell line authentication was performed using SNP array profile or STR profiling for ATCC cell lines. Cells were checked routinely by PCR for the absence of mycoplasma. Cell lines were grown in RPMI 1640 (CLB-GA, CLB-GE, and CLB-MA) or DMEM (IMR-32, SK-N-BE(2)C, and HEK293T) medium supplemented with 10% FBS and antibiotics. ETV5 invalidation was performed with Silencer®Select validated siRNA from Ambion (ThermoFischer Scientific) with the RNAimax transfection reagent (ThermoFischer Scientific). To obtain a total protein lysate, cells were washed in PBS containing 50 µM of sodium orthovanadate and lysed using NP-40 lysis buffer (10 mM Tris-HCl, 5 mM EDTA, 150 mM NaCl, 1% NP-40, 10% glycerol, 20 mM NaF, 1 mM sodium orthovanadate, Complete Protease Inhibitor Cocktail, and PhosSTOP Phosphatase Inhibitor from Roche Diagnostics). Anti-phospho-ALK, anti-ALK, anti-phospho-ERK antibodies were from Cell Signaling Technology (#3341, 3633, and 4370, respectively). Anti-ERK was from Millipore (06-182), anti-ETV5 from Abnova (H00002119-M02), anti-RET from Abcam (ab134100), and anti-actin from Sigma (A-5316). TAE684 and trametinib were purchased from Selleckem. mAb46 is a kind gift from Pr. Marc Vigny.