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. 2017 Feb 15;8:14228. doi: 10.1038/ncomms14228

Figure 3. Dub3 deubiquitinates Snail1 and antagonizes the function of Snail1's E3 ligase.

Figure 3

(a) Flag-Snail1 was co-expressed with vector or Myc-Dub3 in HEK293 cells. After treatment with cycloheximide (CHX) for the indicated time intervals, expression of Snail1 and Dub3 was analysed by western blot (top panel) using Flag and Myc antibodies, respectively. The intensity of Snail1 expression for each time point was quantified by densitometry and plotted (bottom panel). Experiment was repeated three times and a representative experiment is presented (mean±s.e.m. in three separate experiments). (b) MDA-MB231 cells were transfected with control or Dub3 siRNA. After treatment with CHX as indicated above, expression of endogenous Snail1 and Dub3 was analysed by western blot (top panel); the intensity of Snail1 expression for each time point was quantified by densitometry and plotted (bottom panel) (mean±s.e.m. in three separate experiments). Experiment was repeated three times and a representative experiment is presented. (c) Flag-Snail1 and HA-ubiquitin were co-expressed with WT or CS mutant Dub3 in HEK293 cells. After treatment with 10 μM MG132 for 6 hr, Snail1 was subjected to IP and the poly-ubiquitination of Snail1 assessed by western blot using HA antibody. IP Snail1 was blotted using Flag antibody. Input protein levels of Snail1 and Dub3 were examined using Flag and Myc antibodies, respectively. (d) MDA-MB231 and MDA-MB157 cells stably transfected with control, or Dub3 shRNA were treated with MG132 for 6 hr. Extracts were subjected to IP with Snail1 antibody and the poly-ubiquitination of Snail1 assessed by western blot using ubiquitin antibody. Input of Snail1 and Dub3 were analysed by western blot. (e) Ubiquitinated Snail1 was purified from MG132-treated HEK293 cells expressing Flag-Snail1, and then incubated with purified Myc-tagged WT-Dub3 or CS-Dub3 in a deubiquitination assay as described in Experimental Procedures. The poly-ubiquitinated state of Snail1 was assessed by western blot using HA antibody. The immuno-purified Snail1 and Dub3 used in this assay were analysed using Flag and Myc antibodies, respectively. (f) Flag-Snail1 was co-expressed with the indicated expression plasmids, and the expression of Snail1, Dub3, FBXL14, and β-TRCP1 were analysed by western blot. (g) MDA-MB231 cells were transfected with indicated siRNA and cell lysates were analysed by western blot. (h) Flag-Snail1 and HA-ubiquitin were co-expressed with indicated expression plasmids in HEK293 cells. After treatment with 10 μM MG132 for 6 h, Snail1 was obtained by IP and the poly-ubiquitination of Snail1 assessed detected by western blot using HA antibody. IP Snail1 was blotted using Flag antibody. Input protein levels for Dub3, FBXL14 and β-TRCP1 were assessed by western blot.