Molecular Profiling of ALK Signaling

RNA was extracted using RNeasy Mini kit (Qiagen) and quantified using the Nanodrop. qRT-PCR was performed using inventoried TaqMan Gene Expression Assays (Applied Biosystems). RNA expression values were normalised to GAPDH and all experiments were performed in triplicate. Cell lysates were harvested using PhosphoSafe lysis buffer (Merck Millipore Ltd, Feltham, UK) and Western blotting carried out as previously described [39 (link)]. Primary antibodies used were p ALK (Tyr-1604 and Tyr-1282/83), ALK, p ERK, ERK, pAKT, AKT, pSTAT3 (Cell Signaling, Leiden, The Netherlands), STAT3 (R&D Systems) MYCN and GAPDH (Santa Cruz Biotechnology, Heidelberg, Germany), all at 1:1000. Flow cytometry and Caspase 3/7 assays were performed as previously described [39 (link)].