Figure 3. MAGOHB and MAGOH interact with the same proteins and elicit NMD. (A) FLAG-MAGOH and FLAG-MAGOHB were expressed in HeLa cells. Complexes were immunoprecipitated using FLAG-agarose beads. Western blotting with specified antibodies was used to visualize co-precipitated endogenous proteins. (B) Equal amounts of FLAG-Y14 were transfected together with increasing amounts of V5-MAGOHB or V5-MAGOH. Immunoprecipitations were performed using FLAG beads and co-precipitated components of the EJC were detected by immunoblotting. (C) Equal amounts of FLAG-MAGOH and FLAG-MAGOHB were transfected together with increasing amounts of V5-MAGOHB and V5-MAGOH respectively. Immunoprecipitations were performed using FLAG beads and immunoblotting was performed to visualize co-precipitated proteins. (D) A ³²P bodylabeled splicing substrate (MINX RNA) was incubated in HeLa nuclear extracts and HEK293 whole cell extracts expressing FLAG-MAGOHB, FLAG-MAGOH, or FLAG-Y14. FLAG-tagged proteins were immunoprecipitated and co-precipitated RNA was separated by denaturing PAGE. (E) Northern blot of HeLa cells co-transfected with the β-globin 4boxB reporter plasmid, a control and the indicated λNV5-fusion proteins. Percentages represent the mean calculated from three independent experiments.