Notch1 intracellular domain is a Snail binding partner. (A) Expression of the S protein-Flag-SBP (streptavidin-binding peptide) (SFB)-Snail protein in stably transfected cell lines was assessed by immunoblot analysis. (B) Tandem affinity purification of Snail-bound protein complexes was conducted using 293T cells stably expressing SFB-tagged Snail. Bound proteins were separated by SDS-PAGE and examined by silver staining. The Notch1 protein and the number of peptides identified by mass spectrometry are shown in the table at the bottom. "Position" indicates the position of the peptide in the amino acid sequence of Notch1. (C) SFB-Snail-overexpressing cells were treated with 10 μM MG132 for 6 hours and coimmunoprecipitated using anti-Flag and anti-Notch1 antibodies. Immunoblot analysis showed an interaction between Flag-Snail and Notch1 intracellular domain (NICD). Immunoglobulin G (IgG) served as a negative control. (D) Hep3B cells were treated with 10 μM MG132 for 6 hours and coimmunoprecipitated using anti-Snail and anti-Notch1 antibodies. Immunoblot analysis showed an interaction between endogenous Snail and the Notch1 (NICD) protein. IgG served as a negative control. (E) Hep3B cells were immunostained with anti-Snail and/or anti-Notch1 antibodies and assessed using the Duolink II assay. Red foci indicate interactions between endogenous Snail and Notch1 proteins. (F) Hep3B cells were transfected with hemagglutinin (HA)-Snail and Myc-NICD and then stained with anti-HA and anti-Myc antibodies. Cellular localization of Snail (red) and NICD (green) was examined. Nuclei were stained with 4', 6-diamidino-2-phenylindole (DAPI blue).