Fig. 5. Biochemical analysis of the expression and subcellular distribution of cadherins and ß-catenin in response to ectopic Pax3 expression in Saos-2 cells. (A) The turnover rate of ß-catenin protein was assessed by pulse-chase analysis. Control and Ad-Pax3^flag-infected cells were pulse labeled at three days postinfection with S³⁵ methionine and chased in the absence of label for the times indicated. Cell extracts were prepared and equivalent amounts were immunoprecipitated with antibodies directed against ß-catenin. (B) The steady-state levels and distribution between the NP-40 soluble (S) and insoluble (I) fractions at three days postinfection of cadherins and ß-catenin was assessed by immunoblot analysis of equivalent amounts of cell extracts of Ad-ßgal- or Ad-Pax3^flag-infected cells. Pax3 expression resulted in altered distribution of a pan-cadherin product (B, top panel) and ß-catenin (B, third panel) and in a reduction in the total levels of cadherin-11 (B, second panel). Actin immunoblot (B, bottom panel) served as a loading control.