Fig. 4

UCHL3 interacts with and stabilizes CTNNB1. A Immunoprecipitation with anti-Flag. HA-CTNNB1 vectors were coexpressed into 293 T cells with Flag-UCHL3 vectors. B Immunoprecipitation with anti-CTNNB1 followed by immunoblotting with anti-UCHL3 in T24 cells. C Colocalization of HA-CTNNB1 and Flag-UCHL3 in 293 T cells. Bar = 10 µm. D Western blot analysis of the effect of increasing amounts of UCHL3 (0, 200, 400, 600 and 800 ng) on CTNNB1 expression in 293 T cells. E Western blotting with the HA antibody was performed to measure CTNNB1 expression in T24 cells when HA-CTNNB1-overexpressing vectors and Flag-UCHL3-overexpressing vectors or Flag-UCHL3^C94A-overexpressing vectors were cointroduced. F Western blot analysis of CTNNB1 expression in UCHL3-deficient T24 cells. G, H Western blot analysis of CTNNB1 expression stability in the presence or absence of UCHL3 (293 T or T24) after treatment with cycloheximide (CHX) (50 μg/ml) as measured at the indicated time points. I Western blot analysis of CTNNB1 ubiquitination in the presence of Flag-UCHL3 or the Flag-UCHL3^C94A mutant