FIGURE 4 from MARCH2, a Novel Oncogene-regulated SNAIL E3 Ligase, Suppresses Triple-negative Breast Cancer Metastases

MARCH2 promotes ubiquitination and downregulation of SNAIL. A, HA-tagged WT, but not RING domain–mutant (W97A), MARCH2 downregulates SNAIL levels in MDA-MB-231 cells. B, MARCH2 coprecipitates with SNAIL in MDA-MB-231 cells coexpressing FLAG-SNAIL and HA-MARCH2. C, WT, but not RING domain– or transmembrane domain–mutant, MARCH2 ubiquitinates SNAIL. FLAG-Snail was immunoprecipitated from MDA-MB-231 cells coexpressing FLAG-Snail, HA-Ubiquitin and GST-tagged MARCH2 constructs. Immunoprecipitates were blotted with antibodies to HA to assess ubiquitination. D, MARCH2 ubiquitinates SNAIL in in vitro ubiquitination assay. Reactions were performed with recombinant SNAIL (Myc-tagged) in the presence of ubiquitin, recombinant E1 (UBE1), recombinant E2 (UBE2D3) and/or recombinant MARCH2.