Alternative translation sites produce mislocalized MMSET variants. (A) Immunoblot of transiently transfected HeLa cells with various MMSET constructs. GFP-protein indicates N-terminally tagged constructs and protein-GFP indicates C-terminally tagged constructs. The detection of the predicted protein products in the C-terminally tagged MB4-2, MB4-3, and RE-IIBP constructs confirms that the alternative translation initiation sites in exon 4, 6, and 15 are functional. Interestingly, the MB4-2 construct resulted in 2 protein products, indicating that neither alternative translation site is dominant. (B) Live cell localization of type I and II constructs of MB4-2 and MB4-3 MMSET variants C-terminally tagged with GFP in transiently transfected HeLa cells. The live cell permeable DNA stain, Hoechst, is used to identify the nucleus and nucleoli. All 4 novel MMSET constructs, which are unique to the MB4-2 and MB4-3 breakpoint variants, result in proteins that enrich in nucleoli unlike the wild-type variants that are excluded from nucleoli.