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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.
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