Figure 3

Wnt3a stimulates a rapid shuttling of Wnt/β-catenin signaling elements. Panel A, quantified immunoblot analysis of subcellular fractions obtained from control cells (time = 0) and cells stimulated with purified Wnt3a. F9 cells expressing Rfz1 receptor were stimulated with Wnt3a for the indicated times. Cell cultures were collected, disrupted, and fractionated to the plasma membrane, cytoplasm and nuclei fractions, as described in Methods. Each fraction (100 μg) was subjected to SDS-PAGE and analyzed by immunoblotting with specific antibodies targeting the signaling molecules indicated. The three panels displayed at the bottom of the immunoblot set show blots stained with antibodies to well known subcellular marker proteins: Na⁺-K⁺-ATPase (plasma membrane), GAPDH (cytoplasm) and fibrillarin (nuclei), respectively. The result shown is representative of 10 independent experiments. Panel B, summary of the quantified trafficking of signal elements in response to Wnt3a. Bands were quantified by densitometry as described in Methods and values are displayed as fold of zero time point. The content of key signaling molecules in the plasma membrane- (PM, blue line), cytoplasmic- (CY, pink line) and nuclear- (NU, green line) enriched subcellular fractions are displayed. The results are shown as mean values ± S.E. from 6–10 independent experiments.