Fig 3. Interaction of region B with RanGTP. A. Yeast two hybrid screening. Left panel, schematic representation of the bait, the Bt region of Mm_Nemp1 (DBD-Mm_Bt). Right panels, colony formation (in duplicate) of yeast AH109 cells transformed with DBD (upper) or DBD-Mm_Bt (lower) with AD-Mm_Ran on plates lacking tryptophan, leucine, and adenine. DBD, the DNA binding domain of Gal4; AD, the activation domain of Gal4. B. Co-IP of region B with Ran or its mutants using Xenopus embryos. mRNA for HA-tagged Mm_Bt was coinjected into Xenopus embryos with mRNA for a Myc-tagged construct of Mm_Ran, the RanGDP form mutant T24N, the RanGTP form mutant Q69L, or EGFP. Experimental conditions were the same as in Fig 2. C. GST pulldown assays using Xenopus embryo lysates. Purified GST or GST-Mm_Bt protein absorbed onto glutathione-Sepharose beads were incubated with lysates of Xenopus embryos, which had been injected with mRNA for HA-Mm_Ran, HA-T24N, or HA-Myc-Q69L (500 pg/embryo). Proteins bound to the beads were analyzed by western blotting. D. In vitro binding assays with recombinant proteins, Myc-Mm_RanQ69L(GTP) and GST-Mm_Bt. Purified GST-Mm_Bt or GST (2.8 μg) was incubated with purified Myc-RanQ69L (5 μg), which had been loaded with 2 mM GTP in the binding buffer. E. Co-IP of Mm_Ran mutants T42A and δC with Mm_Bt-HA using Xenopus embryos. mRNA for HA-tagged Mm_Bt was coinjected into Xenopus embryos with mRNA for Myc-tagged Mm_Ran, or its mutants (T42A or δC). Unnecessary lanes were removed from a single blot. F. GST pulldown assays of GST-Mm_RanQ69L, importin β and Mm_Bt. Xenopus embryos were injected with mRNA for FLAG-tagged importin β. Lysates were added with 0.1, 1, 10 μg of recombinant Mm_Bt and glutathione beads absorbed 10 μg of GST-RanQ69L. Western blotting was performed with antibodies as indicated below each panel. Arrowheads, expected product bands.
Image published in: Shibano T et al. (2015)
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