Figure 4.

API5 is associated with the mRNA export machineries. (A) Left, disease–function annotations and pathways analyses of the API5 interaction partner list derived from immunoprecipitation/proteomics analysis using the IPA program. Top 7 pathways (P < 10⁻⁹) were shown in magenta. Right, API5 interaction partners that are members of the human TREX or eIF4E/LRPPRC mRNA export complex. API5–FGF2 interaction-dependent API5 interaction partners are shown in red text with green background (‘WT only’ in Table 2). API5–FGF2 interaction-independent API5 interaction partner is shown in blue text (‘All’ in Table 2). The proteins shown in black text are API5 interaction partners partially dependent on API5–FGF2 interaction (‘WT ↑’ in Table 2). ERH and THOC7 (in white text with grey background) which are known members of the TREX complex were not identified positive in our proteomics analysis. (B) Representative mass spectrometry data of identified API5-binding proteins related to the mRNA export machineries. (C) Validation of the interaction between API5 and mRNA export machinery components by immunoprecipitation. API5 knockout (gAPI5) and reconstituted (WT or 4Mut) HeLa cells were used. RNase A was used during immunoprecipitation experiments to exclude any possible RNA-mediated interactions. (D) GST pulldown experiments with purified recombinant GST-UAP56, His-API5 and His-LMW FGF2 to monitor the direct interactions between UAP56 and API5, UAP56 and FGF2, and UAP56 and the API5–FGF2 complex. Protein bands were visualized both by Coomassie blue staining (up) and western blot analysis (down). Due to the contaminant protein bands of similar molecular weight to His-API5 (Marked in an asterisk, *), the His-API5 bands could not be distinguished clearly by Coomassie blue staining. Therefore, the His-API5 and His-FGF2 bands were validated by western blot with Anti-His tag antibody. The multiple bands marked in a double asterisk (**; lanes 3, 4 and 5) are likely to be non-specific bands. (E) Monitoring direct interactions between UAP56 and API5, UAP56 and FGF2, and UAP56 and the API5–FGF2 complex by SPR experiments. The concentrations of each analyte protein are indicated. The SPR experiment with UAP56 and PBK (PDZ-binding kinase) is the negative control (N.B.; no binding).