Supplementary Figure 6: A20-mediated Snail1 monoubiquitination is required for nuclear retention of Snail1 and interaction with transcriptional co-repressors.

(a) A plasmid encoding HA-GSK3β was transfected into HEK293 cells with or without A20 expression plasmid. Cell lysates were immunoprecipitated with anti-A20 antibody and subsequently immunoblotted. (b) NMuMG cells were infected with retroviruses expressing wild-type Snail1 (Flag-Snail1-WT) or the Snail1(3KR) mutant (Flag-Snail1-3KR). After cells were stained with anti-Flag antibody and DAPI, the localization of Snail1 protein was observed by confocal microscopy. Scale bars, 20 μm. (c) β-TrCP1 depletion in NMuMG cells by different siRNAs targeting β-TrCP1 mRNA or control siRNA (siCON) was confirmed by immunoblot analysis with anti-β-TrCP1 antibody. (d) β-TrCP1-depleted (siβTrCP1#2) NMuMG cells were transfected with a plasmid encoding Flag-Snail1-WT or Flag-Snail1-3KR in the absence or presence of HA-A20. Cell lysates were immunoblotted. (e) A20-depleted and control shGFP-expressing NMuMG cells were treated with TGF-β1 (5 ng ml⁻¹) for 24 h, followed by exposure to MG132 (10 μM) for 4 h and fractionated into cytoplasmic and nuclear extracts. Both extracts were immunoblotted with the indicated antibodies. Expressions of tubulin and lamin were used as cytoplamic and nuclear markers, respectively, and loading controls. (f) A plasmid encoding Flag-Snail1-WT or Flag-Snail1-3KR was co-transfected into NMuMG cells with or without HA-A20 plasmid. Cell lysates were immunoprecipitated with anti-Flag antibody and subsequently immunoblotted. (g) Chromatin immunoprecipitation analysis (ChIP) on NMuMG cells transfected with a plasmid encoding Flag-Snail1-WT or Flag-Snail1-3KR. Chromatin fragments were immunoprecipitated with anti-Flag antibody. PCR primers for E-cadherin promoter region were used to amplify the DNA isolated from the immunoprecipated chromatins and input samples. The data in quantitative real-time PCR (lower panel) were statistically analyzed by a t-test and show the mean ± s.d. of n = 3 independent experiments. ^∗∗∗P < 0.01 compared to IgG control. n.s.; not significant. Images shown in this figure are representative of n = 3 independent experiments. Expression of β-actin was used as a loading control for the immunoblot analysis except for e. Statistics source data for g are available in Supplementary Table 3. Unprocessed original scans of blots in a and c–f are in Supplementary Fig. 9.