Fig. 5. 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-NA, 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-XRnf12N (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-Ldb1C. XRnf12 causes degradation of Ldb1C 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-Ldb1C instead of anti-FLAG antibody used in the rest of the experiments in Fig. 5. Amounts of mRNAs injected (ng/embryo): FLAG-Ldb1C, 0.5; XRnf12 constructs, 0.5; Xlim-1 constructs, 2.0.