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. 2019 Jul 30;47(17):9313–9328. doi: 10.1093/nar/gkz643

Figure 4.

Figure 4.

STAU1 promotes an IMPβ-mediated replacement of the CBC by eIF4E in vivo and in vitro. (A) IPs of IMPβ-FLAG in HEK293T cells depleted of STAU1. n = 3. (B–D) The in vitro replacement assay. (B) A schematic diagram of the experimental procedure. For the immunopurification of FLAG-tagged proteins, the extracts of the cells transiently expressing either FLAG-GFP or IMPβ-FLAG were subjected to IPs with anti-FLAG antibody–conjugated agarose beads. The bead-bound FLAG-tagged proteins were eluted by means of 3 × FLAG peptides. For immunopurification of CBC-associated reporter mRNPs, the IPs of endogenous CBP80 were carried out in the extracts of either undepleted or STAU1-depleted cells transiently expressing SL-5′ intron RLuc reporter mRNAs. (C) An in vitro replacement assay using reporter mRNPs immunopurified from the undepleted cells. The bead-bound mRNPs were mixed with either immunopurified FLAG-GFP or IMPβ-FLAG. After the reaction, the bead-bound mRNAs were analyzed by qRT-PCRs. n = 5; *, P < 0.05. (D) An in vitro replacement assay involving reporter mRNPs immunopurified from the STAU1-depleted cells. n = 5; #, not significant.