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A high level of Xlim-1 suppresses XRnf12-mediated degradation of Ldb1 through interaction with Ldb1. (A) Effects of Xlim-1 on XRnf12-mediated degradation of Ldb1. The experimental design is the same as in Fig. 4A,B. Xlim-1 increases the expression level of FLAG-Ldb1 dose-dependently in the presence (lanes 3-7) or absence (lanes 8-12) of XRnf12. Comparison between lanes 6 and 7, and lanes 11 and 12 suggests that Xlim-1 suppresses Ldb1 degradation by XRnf12. See text for details. Amounts of mRNAs (ng/embryo): FLAG-Ldb1, 0.5; XRnf12, 0.25; Xlim-1, 0.25 (lanes 4,9), 0.5 (lanes 5,10), 1.0 (lanes 6,11), 2.0 (lanes 7,12). (B) The LIM domain-containing fragment (ABL60) of Xlim-1 is sufficient for the suppression of XRnf12-mediated degradation of Ldb1. A series of Xlim-1 constructs depicted in E were tested at the highest dose used in A (lanes 7,12) for their ability to block Ldb1 degradation by XRnf12. ABL60, which contains the LIM domains, efficiently blocks Ldb1 degradation whereas other constructs does not. Amounts of mRNAs (ng/embryo): FLAG-Ldb1, 0.5; XRnf12, 0.25; Xlim-1 constructs, 2.0. (C) LIM-only protein LMO2 also efficiently blocks Ldb1 degradation in a different set of experiments designed as in B. (D) Interactions between 35S-labeled XRnf12 and a series of GST-Xlim-1 constructs depicted in E were analyzed by GST pull-down assay. XRnf12 shows weak interactions with GST-HD27 and GST-ΔNA, while other GST-Xlim-1 constructs shows little or no interaction with XRnf12. GST-Ldb1 shows stronger interaction with XRnf12 than GST-Xlim-1 does. XRnf12 also shows weak self-interaction with GST-XRnf12ΔN (aa 282-616). GST alone serves as a negative control. Coomassie brilliant blue staining (lower panel) shows comparable amounts of GST fusion proteins (indicated by dots) used in the assay. (E) Representation of the GST-Xlim-1 constructs used for mapping experiments and the summary of the results shown in D. The homeodomain-containing region (aa 178-265) of Xlim-1 is necessary and sufficient for the interaction with XRnf12. A, B, LIM domains A and B; HD, homeodomain; n.d., not done; numbers, amino acid positions. (F) The LIM interaction domain of Ldb1 is required for the suppression of XRnf12-mediated Ldb1 degradation by Xlim-1 as revealed by the use of FLAG-Ldb1ΔC. XRnf12 causes degradation of Ldb1ΔC in a RING-dependent manner, which is not suppressed by Xlim-1 or ABL60. To avoid an overlap with a non-specific band, we used anti-Ldb1/CLIM2 (N-18) antibody in F to detect FLAG-Ldb1ΔC instead of anti-FLAG antibody used in the rest of the experiments in Fig. 5. Amounts of mRNAs injected (ng/embryo): FLAG-Ldb1ΔC, 0.5; XRnf12 constructs, 0.5; Xlim-1 constructs, 2.0. Image published in: Hiratani I et al. (2003) Copyright © 2003. Image reproduced with permission of the publisher 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

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