Fig. 1.

Slug interacts with PRMT5 and LSD1. (A) Immunoaffinity purification and mass spectrometry analysis of Slug-binding proteins. Extracts from HEK293T cells bearing Flag (Vector) or Flag-Slug were immunopurified with anti-Flag affinity columns and eluted with Flag peptide. The eluates were resolved by SDS-PAGE and visualized by silver staining. The protein bands on the gel were excised and identified by mass spectrometry. Representative peptide fragments of PRMT5 and LSD1 are indicated on the right. (B) Representative peptide coverage of the indicated proteins is shown in the table. (C) The purified fractions were analysed by western blotting with antibodies against indicated proteins. (D) Cell lysates from MDA-MB-231, SUM159 or Hs578T cells were immunoprecipitated with antibodies against indicated proteins followed by immunoblotting with various antibodies indicated. The arrows denote the light chains of IgG and Slug antibody. The asterisks indicate a nonspecific band. (E) Flag-PRMT5, HA-Slug and HA-LSD1 (Left), Flag-Slug, HA-PRMT5 and HA-LSD1 (Middle) or Flag-LSD1, HA-Slug and HA-PRMT5 (Right) were co-expressed in HEK293T cells, respectively. After immunoprecipitation with appropriate antibodies, bound proteins (e.g., Slug, PRMT5 or LSD1) were examined by western blotting. (F) GST-fused Slug, LSD1 or PRMT5 were incubated with the Flag-tagged PRMT5, Slug or LSD1 purified from HEK293T cells. The binding proteins by GST pull-down assays were examined by western blotting with indicated antibodies. Coomassie brilliant blue staining of the GST-fused proteins was shown below