Figure 2. Dub3 interacts with Snail1.

(a) HA-Snail1 was co-expressed with vector or Flag-Dub3 in HEK293 cells. Snail1 and Dub3 were immunoprecipitated (IP) with HA or Flag antibody, respectively, and the associated Dub3 and Snail1 were analysed by western blot using either Flag or HA antibody. One-fortieth of the lysate from each sample was subjected to western blot to examine the expression of Snail1 and Dub3 (input lysate). (b) Endogenous Snail1 and Dub3 were captured by IP from MDA-MB231 and MDA-MB157 cells, and the bound endogenous Dub3 and Snail1 were examined by western blot. (c) Schematic diagram showing the structure of Snail1 and deletion constructs used (top panel). Flag-tagged full-length (FL) or deletion mutants of Snail1 were co-expressed with Myc-Dub3 in HEK293 cells. Extracts were subjected to IP with Flag or Myc antibody, and bound Dub3 or Snail1 was analysed by Western blot using either Myc or Flag antibody. (d) Lysates from HEK293 cells expressing WT or different deletion mutants of Flag-Snail1 were mixed with GST-Dub3. After pull-down by glutathione-agarose, the associated proteins were analysed by western blot. (e) Schematic diagram showing the structure of Dub3 and deletion constructs used (top panel). Myc-tagged full-length or deletion mutants of Dub3 were co-expressed HA-Snail1 in HEK293 cells. Extracts were subjected to IP with Myc or HA antibody, and the bound Snail1 or Dub3 was analysed by western blot using either HA or Myc antibody. (f) Lysates from HEK293 cells expressing WT or different deletion mutants of Myc-Dub3 were mixed with GST-Snail1. After pull-down by glutathione-agarose, the associated proteins were analysed by western blot. (g) Myc-Dub3 was co-expressed with Flag-tagged full-length or deletion mutants of Snail1 in HEK293 cells. The protein expressions of Dub3 and Snail1 were analysed by western blot. (h) Endogenous Dub3 and Snail1 in MDA-MB231 cells was detected by IF staining. Scale bars, 20 μm.