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. 2011 Mar 16;19(8):875–881. doi: 10.1038/ejhg.2011.42

Figure 3.

Figure 3

Functional studies with mutant LRP5 protein. (a) Dual-luciferase reporter assay measuring the canonical Wnt signaling activity. The co-transfected Firefly luciferase reportergen construct (Topflash TCF reporter plasmid) contains TCF-binding sites as a target for activated canonical Wnt signaling. The graph describes the relative luciferase activities (average and standard deviation) of empty vector (pcDNA3.1), wild-type LRP5 (LRP5-WT9L), and the mutant LRP5-p.E528_V529ins21. The mutant LRP5-Mut3L serves as a positive control for impaired Wnt/Norrin signal transduction.25 Note that activities of the mutant LRP5-p.E528_V529ins21 are significantly lower than those of LRP5-WT9 L (P-values <0.001 (***) in a two-sided Student's t-test calculation). (b) Western blot of cell lysates from transiently transfected HEK293T cells used in the dual-luciferase reporter assay (results presented in a), confirming expression of WT9L and of the two mutated LRP5 receptors. (c) Cell fractioning assay. HEK293T cells were transiently transfected with LRP5-WT9L or LRP5-p.E528_V529ins21. Both WT9L and p.E528_V529ins21 are almost exclusively detectable in the membrane fraction. (d) Secretion assay. Representative western blot of conditioned medium (CM) and cell lysate (LY) from HEK293T cells transiently transfected with LRP5N-WT9L, LRP5N-p.E528_V529ins21 and LRP5N-Mut3L expression constructs. Comparable loading of cell lysate or conditioned medium across experiments is indicated by immunodetection of β-actin or by Coomassie staining of a 60-kDa protein in the conditioned medium, respectively. Note that only LRP5N-WT9L is strongly detected in conditioned medium.