Figure 3: DUB3 deubiquitinates and stabilizes SNAIL1.

(a) Cell extracts were prepared from four luminal- and four basal-like subtypes of human breast cancer cell lines, and expression of SNAIL1, DUB3, E-cadherin and vimentin was analysed by western blotting. (b) MDA-MB-231 cells stably expressing control or DUB3 shRNAs were generated and western blot was performed with the indicated antibodies. (c) Total RNA was isolated from cells in b. Relative expression of SNAIL1 in cells stably expressing control or DUB3 shRNAs was determined by quantitative PCR. Transcript levels were determined relative to actin mRNA levels and normalized relative to control cells. The results represent the means (±s.d.) of three independent experiments. (d) MDA-MB-231 cells stably expressing control or DUB3 shRNAs were treated with vehicle or MG-132 and western blot was performed with the indicated antibodies. (e) T47D and MCF-7 were infected with virus containing vector, FLAG-DUB3 and the C89S mutant and western blot was performed. (f) Cycloheximide pulse-chase assay was performed in cells as in b and results are quantified in g. The results represent the means (±s.d.) of three independent experiments. ^##P<0.01. (h) Cells were cotransfected with indicated plasmids and treated with MG-132 for 6 h before cell lysates were boiled and immunoprecipitated with HA beads, and the polyubiquitylated SNAIL1 protein was detected by anti-ubiquitin antibody. (i) Cells were transfected with HA-SNAIL1 and treated with MG-132 for 6 h. Cell lysates were immunoprecipitated with HA beads and incubated with GST, GST-DUB3 or GST-DUB3 C89S mutant in a cell-free condition. The polyubiquitylated SNAIL1 protein was detected by anti-ubiquitin antibody. (j) MDA-MB-231 cells stably expressing control or DUB3 shRNAs were transfected with indicated constructs and Ni-NTA beads were used to pull down His-tagged ubiquitin, and the polyubiquitylated SNAIL1 protein was examined.