UCH L1 up-regulates β-catenin signaling.

<p>A. Reduction of expression of UCH L1 correlates with decrease in β-catenin protein levels. 293 cells were transfected with control GFP siRNA or two UCH L1 siRNAs in pRS vector and maintained in medium containing puromycin. After 3 weeks of selection total lysates from control- and si1/2 UCH L1-transfected cells were resolved in 12% PAGE, transferred to PVDF membrane and probed with β-catenin and UCH L1 antibodies. B. More β-catenin accumulates in UCH L1 siRNA-expressing cells in the presence of proteasome inhibitor. Stable 293 cell lines expressing control and UCH L1 siRNAs were treated for 4 h with 25 mM MG101 or DMSO as negative control. β-catenin was immunoprecicpitated from total lysates, IPs were resolved in 10% PAGE and probed with β-catenin antibodies. Mouse normal immunoglobulins were used as control for IPs. Arrow heads indicate ubiquitinated forms of β-catenin. C. UCH L1 is involved in deubiquitination of β-catenin <i>in vitro</i>. β-catenin was <i>in vitro</i>-translated (lane 4) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005955#s3" target="_blank">Material and Methods</a>. To induce further ubiquitination equal amounts of translated β-catenin were pre-incubated with Ub mix for 30 min, and an <i>in-vitro</i> deubiquitination reaction was carried out in the absence (lane 1) or presence of 2 or 4 mM recombinant UCH L1 (lanes 2 and 3). Western blot was performed with β-catenin antibody. D. Deubiquitinating activity of UCH L1 is required for TCF4 transcriptional activity. UCH L1 wild type or UCH L1 inactive mutants C90S and H161D were co-transfected with either TOPFlash or FOPFlash into 293 and 3T3 cells. Luciferase activity and expression of HA-UCH L1 proteins were determined 48 h post-transfection. Where indicated, cells were treated with 25 mM LiCl 6 h before harvesting. The data represent two independent experiments prepared in triplicate and normalized to FOPFlash activity.