Figure 4. The SNAG domain is absolutely required for Snail2-mediated EMT while the SLUG domain has a negative modulatory action.

Snail2-HA wild type and the Snail2-ΔSNAG or the Snail2-ΔSLUG mutants were stably transfected into MDCK cells and selected clones analyzed for phenotype and EMT markers. (A) Phenotypic characterization of one representative Snail2-HA clone (#3), two representative Snail2-ΔSNAG clones (#2 and #3) and two representative Snail2-ΔSLUG clones (#1 and #3) compared to control MDCK-CMV (CMV) cells. Panels a to f, phase contrast images; g to l, immunofluorescence staining for E-cadherin (green). Nuclei are stained with DAPI (blue). Bars, 200 µm. (B) Western blot analysis of the indicated clones for E-cadherin (upper) and vimentin (middle); migration of the indicated proteins is shown in kDa at the left. (C) Western blot analysis of the indicated clones for Snail2-HA, wt or mutants, detection using anti-HA antibodies. Migration of Snail2-HA wild type (32 kDa) and the ΔSLUG mutant (26 kDa) is indicated. α -tubulin (55 kDa) was used as loading control in (B) and (C). Similar results were obtained for all isolated clones. (D) The stability of Snail2-HA and the indicated Snail2-HA mutants in HEK293T cells was determined by incubation in the presence of the translational inhibitor cycloheximide for the indicated time periods. Upper, Western blot analyses of Snail2-HA (wt and mutants) levels of one representative experiment with anti-HA antibodies; α-tubulin was used as a loading control. Bottom, densitometric quantifications of the relative amount of Snail2-HA and the indicated mutants at the indicated time points. Results show the mean of three independent experiments +/− s.d.