Figure 2.

MYCN stabilization and Myc-nick production by each NCYM variant. (A) NCYM amino acid sequences. Purple, variants found in chimpanzees; red, variants found in humans; green, D90N, a mutant of NCYM in which the 90th aspartic acid is substituted with asparagine. (B) Western blotting of HA-NCYM, MYCN proteins in NCYM SNP plasmid-transfected SH-SY5Y cells. At 24 h after transfection, the cells were subjected to western blotting. Actin was used as a loading control. Control: empty vector. (C) Quantitative real-time RT-PCR analyses of MYCN in NCYM SNP plasmid-transfected SH-SY5Y cells. At 24 h after transfection, mRNA expression levels were measured by real-time RT-PCR with β-actin as an internal control. N.S., not significant. Data were analyzed using Student’s t test (comparison with control). (D) Western blotting of MYCN and Myc-nick proteins in NCYM SNP plasmid-transfected SH-SY5Y cells. At 72 h after transfection, cells were subjected to western blotting. Actin was used as a loading control. (E) Quantification of western blotting analysis of NCYM SNP plasmid-transfected SH-SY5Y cells. At 72 h after transfection, the cells were subjected to western blotting. Myc-nick level was normalized to MYCN level. Data are shown as plots and means of three independent experiments. *p < 0.05. Data were analyzed using Student’s t test (comparison with control).