Af3639

ChIP assay was performed according to the protocol described by Nowak et al.^{60 (link)} with some modifications. Cells (4–6 × 10⁶) were cross-linked with 1% formaldehyde for 15 min at room temperature, then lysed in L1 buffer (50 mM Tris-HCl, pH 8.0, 2 mM EDTA, 0.1% IGEPAL, 10% glycerol, 1 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride, and protease inhibitor cocktail) on ice. Nuclei were pelleted by centrifugation and resuspended in ChIP lysis buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 1% SDS). Chromatin was subjected to sonication and then immunoprecipitated with 2 µg of Snail antibody (AF3639, R&D Systems, Minneapolis, MN) or an irrelevant immunoglobulin G (IgG) overnight, followed by incubation with a 50% slurry of protein G sepharose/salmon sperm DNA (Invitrogen) for 3 h at 4 °C. Bound DNA–protein complexes were eluted, and crosslinks were reversed after a series of washes. Purified DNA was resuspended in TE buffer (10 mM Tris–HCl, pH 8.0, 1 mM EDTA) for PCR analysis. The primers used for the 4E-BP1 and E-cadherin promoters are listed in Supplementary Table 4.

Antibody against human TEL2 (Dilution, 1:1000) was obtained from Sigma (HPA029033). Antibodies against human Snail were obtained from R&D (AF3639) for chromatin immunoprecipitation (ChIP) assays and from Cell Signaling Technology (#3895) for Western blotting. Anti-E-cadherin antibody was obtained from BD Company. Antibodies against HA and Tubulin were obtained from Cell Signaling Technology. Antibody against human SERPINE1 was obtained from Santa Cruz (sc-5297).