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To verify at protein level that Wnt3a-CM indeed stimulates β-catenin dependent Wnt signaling and the effect of Wnt5b overexpression, β-catenin quantification in cytoplasmatic and nuclear protein extracts was performed a day 0. Wnt3a-CM resulted in a threefold increase in nuclear β-catenin when compared to control medium. On protein level, at day 0, 28% β-catenin was observed in 3T3-L1 ARQ197 incubated in Wnt3a-CM and transfected with empty vector. In 3T3-L1 cells overexpressing Wnt5b and incubated with Wnt3a-CM, nuclear β-catenin was 17%, while cells incubated with control medium, transfected with Wnt5b overexpression construct or empty vector, nuclear β-catenin was, respectively, 12% and 10% ( Fig. 4.). Wnt5b overexpression in cells incubated in WNT3a-CM results in a ～65% reduction in β-catenin translocation, but is still ～35% higher than higher than in cells incubated with control medium.
Fig. 4.
Wnt5b overexpression reduces translocation of β-catenin to the nucleus. 3T3-L1 cells were transfected with pSPORT-Wnt5b or pcDNA3.1 empty vector and after 16 h incubation with 20% Wnt3a-CM, nuclear and cytoplasmic proteins were isolated, loaded on SDS–PAGE gel, and incubated with anti-β-catenin. β-catenin level was normalized to the level of β-actin for loading. ?p