Supplementary Figure 3: A20 induces monoubiquitination of the Snail1 protein through ZnF7 domain. | Nature Cell Biology

Supplementary Figure 3: A20 induces monoubiquitination of the Snail1 protein through ZnF7 domain.

From: A20 promotes metastasis of aggressive basal-like breast cancers through multi-monoubiquitylation of Snail1

Supplementary Figure 3

(a) Plasmids encoding Flag-Snail1 and wild type His-Ub were co-transfected with HA-A20, HA-GSK3β and HA-βTrCP1 into NMuMG cells in the indicated combinations. Ni-NTA-mediated pull-down assays were performed and ubiquitinated Snail1 was observed by immunoblotting using anti-Flag antibody. Total cell lysates (TCL) were immunoblotted with the indicated antibodies. (b) Dynamics of the interaction between endogenous A20 and Snail1 in NMuMG cells. Cells were treated with TGF-β1 (5 ng ml−1) for the indicated times, immunoprecitated with anti-Snail1 antibody and immunoblotted with the indicated antibodies. (c) Plasmid encoding wild-type HA-A20 or A20 ZnF7 mutant (HA-A20_ZnF7∗) was co-transfected into HEK293 cells together with Flag-Snail1 plasmid. Cell lysates were immunoprecipitated with anti-HA antibody and subsequently immunoblotted with the indicated antibodies. (d) For in vitro ubiquitination assays, Flag-Snail1 proteins were eluted from HEK293 cells transfected with Flag-Snail1 plasmid, and wild-type GST-A20 and mutant GST-A20_ZnF7∗ proteins were purified from E.coli. The reactions were performed in the indicated combinations and samples were immunoblotted with the indicated antibodies. (e) Plasmids encoding Flag-Snail1 and wild type His-Ub were co-transfected into NMuMG cells with HA-A20. After cells were treated with the ubiquitin isopeptidase inhibitor G5 for 6 h, Ni-NTA pull-down assays were performed, followed by immunoblotting with the indicated antibodies. (f) Plasmid encoding HA-A20 or HA-A20(C103A) mutant was co-transfected into NMuMG cells with HA-GSK3β and HA-βTrCP1 in the presence of His-Ub and Flag-Snail1. After cells were pre-treated with MG132, Ni-NTA pull-down and immunoblot assays were performed. Expression of β-actin was used as a loading control in all immunoblot assays except for d. Immunoblot images in this figure are representative of n = 3 independent experiments. Unprocessed original scans of blots in Supplementary Fig. 3 are in Supplementary Fig. 9.

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