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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) Image reproduced on Xenbase with permission of the publisher and the copyright holder. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution license Larger Image Printer Friendly View |