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Fig. 3. Activation of canonical Wnt signalling by ectopic expression of Dvl-2 in high- and low-level expression systems. (A) HEK293 cells were transiently transfected (`high-level ectopic expression system') with either TCF-dependent transcription responsive (`canonical' Wnt signalling) reporter plasmids, reporter plasmids plus HA-Dvl-2 or reporter plasmids plus HA-Dvl-2-ER. Cultures were re-fed with medium containing 1 µM estradiol or control medium 24 hours prior to analysis for luciferase activity and protein levels in the detergent soluble and insoluble fractions of the cell extracts. (B) Time course of activation of canonical Wnt signalling pathway in Dvl-2-ER single-copy cell line. The cells were re-fed with medium containing 1 µM estradiol or with control medium and then harvested after 10, 30 and 60 minutes (10m/30m/60m) and 24 hours (24h) of induction. Detergent-soluble and -insoluble fractions were analysed by SDS-PAGE and western blotting. Identical protein levels were run on the gels. Blots were probed for the HA-tagged exogenous Dishevelled, for endogenous and exogenous Dvl-2 with a rabbit polyclonal anti-Dvl-2 and for endogenous ß-catenin and GSK-3ß. Note that although GSK-3ß levels remain constant, there was an increase in soluble ß-catenin levels from 30 minutes after ß-estradiol treatment. Also, note the disappearance of the endogenous Dvl-2 protein (anti-Dvl-2 probe, 98kD band) from the soluble fraction following ß-estradiol treatment, paralleling that of the single-copy fusion protein (150 kDa band). Finally, note the similarity in expression levels of the endogenous and exogenous proteins (estimated at three- to fivefold higher for the fusion protein). (C) Analysis of ß-catenin and endogenous Dvl-2 expression in the parental cell line after 60 minutes (60m) and 24 hours (24h) of ß-estradiol treatment. Note that there was no change in expression levels.
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