Cells were lysed with lysis buffer (50 mM Tris-HCl, pH 8.0; 150 mM NaCl, 0.1% NP-40, 0.5% CHAPS) containing 0.1% protease inhibitor cocktail III (Calbiochem, San Diego, CA). The lysates were electrophoretically separated, blotted onto a nitrocellulose membrane and blocked with 4% BlockAce solution (Dainippon Pharmaceutical, Osaka, Japan) for 1 h. The blots were then incubated with antibodies against the following proteins: BIG3 (ref. 21 (link)) (1:200); PHB2 (1:500), NcoR (1:500) and ERα (phospho Y537; 1:500; Abcam, Cambridge, UK); SRC-1 (128E7; 1:500), Shc (1:500), α/β-tubulin (1:1,000), Akt (1:1,000), phospho-Akt (S473; 587F11; 1:1,000), p44/42 MAPK (1:500), phospho-p44/42 MAPK (T202/Y204; 1:500) and phospho-ERα (S104/S106; 1:500; Cell Signaling Technology, Danvers, MA); HDAC1 (H-11; 1:500), IGF-1Rβ (1:500), PI3-kinase p85α (U13; 1:500), phospho-ERα (S118; 1:500), phospho-ERα (S167; 1:500) and laminin B1 (1:100; Santa Cruz Biotechnology, Santa Cruz, CA); phospho-ERα (S305; 1:500; Millipore, Billerica, MA); phosphotyrosine (1:500; Zymed, San Francisco, CA); β-actin (AC-15; 1:5,000) and FLAG-tag M2 (1:5,000; Sigma, St Louis, MO); and HA-tag (1:3,000; Roche, Mannheim, Germany). After incubation with an horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, dilution 1:5,000) or monoclonal anti-rabbit immunoglobulins-peroxidase antibody (RG-16, Sigma, dilition 1:5,000) for 1 h, the blots were developed with an enhanced chemiluminescence system (GE Healthcare, Buckinghamshire, UK) and were scanned using an Image Reader LAS-3000 mini (Fujifilm, Tokyo, Japan). All experiments were performed more than three times in triplicate. Finally, the phosphorylation levels of IGF-1Rβ, Shc, PI3K, Akt, p42/44 MAPK and ERα were assessed through densitometric analysis of immunoblot results using an Image Reader LAS-3000 mini51 . Full-length images of immunoblots are shown in Supplementary Fig. S9.