Fig. 4. Cullin-1, Cul1-C75 and Cul1-C477 decrease ß-catenin ubiquitination in cell lines. H293T cells were transfected with Myc-His-Ubiquitin, HA-ß-catenin and either Cul1-C477, Cullin-1 or Cul1-C75. After 36 hours, 10 µM MG132 (a proteasome inhibitor) was added for 12 hours to accumulate ubiquitinated protein. All ubiquitinated protein was immunoprecipitated (IP) via Myc. Ubiquitinated ß-catenin was visualised with an anti-HA antibody. Co-expression of any of the Cullin-1 gene constructs led to a reduction of the levels of ubiquitinated ß-catenin. The severity of reduction corresponded to the amount of truncation, with the most truncated construct Cul1-C477 causing the most severe reduction (lane 3 IP), the Cul1-C75 (lane5) causing a strong reduction and full-length Cullin-1 (lane 4) causing some reduction. This suggests that Cullin1, Cul1-C75 and Cul1-C477 inhibit ß-catenin ubiquitination, probably by inhibiting the endogenous SCF complex. As a control, 10% of the lysate was removed previous to the IP and analysed for total levels of ß-catenin by anti-HA staining. Co-transfection of Cullin-1 led to increased ß-catenin levels in the lysate control in 4/6 experiments (not shown here). An increase in total ß-catenin is not always detected in these experiments because of the high levels of exogenous ß-catenin and because the ubiquitinated form is only a fraction of the total ß-catenin.