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. 2013 Oct 22;109(12):3084–3091. doi: 10.1038/bjc.2013.653

Figure 1.

Figure 1

Expression analysis of ALK in RMS cell lines. (A) Expression of ALK mRNA in tumour cell lines, using primers spanning the extracellular region (ALK-1) and the intracytoplasmic portion (ALK-2) of ALK gene. β2-Microglobulin (B2M) was used as housekeeping gene. RMS-t, PAX3/7-FOXO1-positive RMS; RMS-not, PAX3/7-FOXO1-negative RMS; NB, neuroblastoma; SKM, fetal skeletal muscle; ALCL, anaplastic large cell lymphoma. (B) Relative ALK mRNA expression levels in RMS-t and RMS-not cell lines measured by qRT–PCR. LAN5 and SKM were included as positive and negative controls, respectively. Distribution of data is represented by box plot analysis (dashed box). *P<0.05. (C) ALK protein expression in RMS-t and -not cell lines by western blotting, using β-actin as protein-loading control. (D) Total (α-ALK, upper panel) and phosphorylated (α-pALK, lower panel) ALK proteins detected in RMS-t and RMS-not cell lines after immunoprecipitation (IP:ALK). LAN5 and KARSPAS-299 (K299) cells were used as positive controls for ALK and NPM-ALK expression, respectively. LAN5 total lysates were used as input (Tot.lys.). Asterisk marks non-specific bands in RMS and NB cells. (E) ALK tyrosine phosphorylation in RH30 and RD cell lines exposed to pleiotrophin (PTN) or ALK agonist antibody (mAb) in the presence (+) or absence (−) of ALK inhibitor crizotinib. Total and phosphorylated ALK and ERK1/2 proteins were detected by western blotting. γ-Tubulin was included as loading control.