Figure 2

Cellular distribution of Wnt/β-catenin signaling elements. Large-scale cultures of mouse F9 cells expressing Rfz1 were harvested and the cells disrupted. Subcellular fractions were prepared from the cell masses and the fractions probed for enrichment in plasma membrane (PM), cytoplasm (CY), and nuclei (NU) as described in detail in the Methods. Samples of the whole-cell homogenate were subjected to subcellular fractionation and their complements of cellular proteins to SDS-PAGE. Panel A, the distribution of well-known marker proteins for subcellular fractions (i.e., Na⁺-K⁺-ATPase as a marker for plasma membrane, GAPDH as a marker for cytoplasm, and fibrillarin as a marker for nuclei) also was analyzed in each fraction to establish purity/enrichment of markers in these fractions. Panel B, resolved proteins were analyzed by immunoblotting, stained with one of the following antibodies: anti-Axin, anti-β-catenin, anti-Dvl2, anti-GSK 3β, anti-p-Ser (9)-GSK 3β, and anti-PP2A C-subunit. The relative amounts of the proteins distributed in each subcellular fraction was established densitometrically, based upon their distribution (%), the total protein content of the homogenate and fractions, and quantified analysis of blots from SDS-PAGE. The results are shown as mean values ± S.E. from 8-10 independent experiments.