Supplementary Figure 4: A20 monoubiquitinates three Snail1 lysine residues, which are crucial for Snail1 stability and TGF-β1-induced EMT.

(a) Plasmids encoding wild type Snail1(Flag-Snail1-WT) or Snail1 mutants (Flag-Snail1-N-6KR and Flag-Snail1-C-8KR) were co-transfected into NMuMG cells with wild-type His-Ub and HA-A20 plasmids in the indicated combinations. Ni-NTA-mediated pull-down assays were performed and ubiquitinated Snail1 was observed by immunoblot analysis using anti-Flag antibody. Total cell lysates (TCL) were immunoblotted with the indicated antibodies. (b) A plasmid encoding wild-type Snail1 (Flag-Snail1) or single K-to-R mutants of Snail1 was co-transfected into NMuMG cells in the absence or presence of HA-A20. Cell lysates were immunoblotted with the indicated antibodies. (c) To examine whether A20-mediated monoubiquitination of Snail1 is linked to the phosphorylation of Snail1 by ERK, a plasmid encoding a Snail1 mutant [Flag-Snail1(S82A/S104A)] or wild-type Snail1, was co-transfected into NMuMG cells with or without HA-A20. Cell lysates were immunoblotted with the indicated antibodies. (d) Snail1 depletion in NMuMG cells by lentiviruses expressing different shRNAs was confirmed by immunoblot analysis with anti-Snail1 antibody. (e) Snail1-depleted NMuMG cells were infected with retroviruses expressing wild-type Snail1 (Flag-Snail1-WT) or the Snail1(3KR) mutant (Flag-Snail1-3KR). After treatment with TGF-β1 (5 ng ml⁻¹) for 48 h to induce EMT, cell lysates were immunoblotted with the indicated antibodies. (f) The CDH1-Luc reporter plasmid was co-transfected into Snail1-depleted NMuMG cells with an indicated plasmid. After treatment with TGF-β1 for 48 h, luciferase activities were measured and normalized. The data were statistically analyzed by a t-test and show the mean ± s.d. of n = 3 independent experiments. ^∗∗P < 0.01 compared to cells not treated with TGF-β1 in the case of shGFP and compared to cells treated with TGF-β1 in others. Immunoblot images in this figure are representative of n = 3 independent experiments and expression of β-actin was used as a loading control. Statistics source data for f are available in Supplementary Table 3. Unprocessed original scans of blots in a–e are in Supplementary Fig. 9.