Figure 4

Relative ratios of β-catenin destruction complex components are altered in Usp9x ^−/Y neocortices. (A–C) Usp9x is part of the β-catenin destruction complex. Endogenous USP9X co-immunoprecipitated from HEK293 lysate along with components of the β-catenin destruction complex using Axin antibody (A) and APC antibody (B). (C) Usp9x immunoprecipitated by Axin antibody from E14.5 Usp9x ^+/Y cortical lysate along with other components of the destruction complex. (D) Immunoblot analysis of β-TrCP protein levels in Usp9x ^+/Y and Usp9x ^−/Y neocortices at E12.5 and E14.5 (n = 3). (E) Quantitation of (D) revealed no difference in total β-TrCP protein levels between Usp9x ^+/Y and Usp9x ^−/Y neocortices at E12.5 or E14.5. (F) Components of destruction complex were co-immunoprecipitated by anti-APC antibody from E14.5 Usp9x ^+/Y and Usp9x ^−/Y cortical lysate. Relatively more β-TrCP and GSK-3β were immunoprecipitated from Usp9x ^−/Y compared to Usp9x ^+/Y. (G) Co-immunoprecipitation of destruction complex components using anti-β-TrCP antibody. Increased GSK-3β protein level co-immunoprecipitated from Usp9x ^−/Y cortical lysate confirming increased interaction with β-TrCP.