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Fig. 2. Mesd binds to mature LRP5/6 but not significantly to other members of the LDLR family. (A) Binding of ¹²⁵I-Mesd (5 nM) to HEK293 cells transient transfected with human HA-tagged LDLR, Myc-tagged LRP5, Myc-tagged LRP6 or control vector. Lower panel, western blot analysis for the expression of the LDLR, LRP5 and LRP6. Equal amounts of cell lysate were applied for each lane. (B) Binding of ¹²⁵I-Mesd (5 nM) to LRP-null CHO cells stably transfected with LRP minireceptor mLRP4, LRP1B minireceptor mLRP1B4, VLDLR, apoER2 or empty pcDNA3 vector only. (C) Binding of ¹²⁵I-Mesd (5 nM) to wild-type murine embryonic fibroblasts (MEF-1) or MEF cell lines genetically deficient in LRP (MEF-2), LDLR (MEF-3) or both (MEF-4). Lower panel, western blot analysis of LRP and the LDLR expression in MEF cell lines. (D) Binding of ¹²⁵I-Mesd (5 nM) to human breast cancer cell line MCF-7, human glioblastoma cell line U87 and human aortic smooth muscle cells (SMC). Lower panel, western blot analysis of LRP expression in these cell lines. Assays were carried out for 4 hours at 4°C in the absence (total) or presence of 500 nM Mesd. Values are the means of triple determinations with the s.d. indicated by error bars. ^*P<0.01 indicates a significant difference compared with control cells transfected with empty pcDNA3 vector.

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