Fig. 3: Characterization of SNRNP70::ALK fusion protein.

a Anti-ALK immunoblotting detects a SNRNP70::ALK fusion protein with a size of approximately 85 kDa (arrow) in the patient tumor. In addition, endogenous full-length ALK proteins (indicated with arrowheads) was detected in the tumor sample. Lysates from SK-N-AS, SK-N-BE(2), CLB-GAR (ALK-R1275Q) and NB1 (ALK-amplified) neuroblastoma cells lines²⁹ were loaded as controls (SK-N-AS and SK-N-BE(2) are ALK negative, while CLB-GAR and NB1 are ALK positive). Anti-pALK immunoblotting detects activation of SNRNP70-ALK fusion protein, and anti-pERK1/2 and pAkt confirm downstream AKT and ERK1/2 activity in the patient tumor. Tubulin was used as loading control. b Both anti-ALK and anti-SNRNP70 detect the approximately 85 kDa SNRNP70::ALK fusion protein in the patient tumor. As controls, SK-N-AS cells expressing either vector control, SNRNP7::ALK, SNRNP70::ALK (I1250T)-3xFLAG, or EML4::ALK V3a constructs were employed. Anti-SNRNP70 immunoblotting also detected endogenous SNRNP70 proteins (indicated with *) in all samples. Arrowheads indicate full-length and cleaved ALK. Arrow indicates the SNRNP70::ALK fusion protein. Note: due to a 3x FLAG tag, the molecular weight of the I1250T mutant SNRNP70::ALK fusion protein is slightly bigger than that of the wildtype fusion one. Original immunoblot figures are shown as Fig. S5. c Detection of active receptor tyrosine kinases by Human RTK array. A proteome Profiler Human Phospho-RTK Array Kit (ARY001B, R&D Systems) was used to detect active receptor tyrosine kinases (RTKs) in the tumor lysate following the protocol provided by the manufacturer. Approximately 300 µg clarified lysate was applied to one array. The layout of different RTKs (purple texts) in the array is provided on top. Boxed dots indicate pALK.