The anti-BIG3 monoclonal antibody was generated by Sigma-Aldrich (Tokyo, Japan). Briefly, a rat was immunised with purified His-tagged human BIG3 protein (459-572 aa). The iliac lymph nodes were collected and fused with myeloma cells, resulting in the formation of a hybridoma. Immunoblot analyses were performed as described previously9 (link). After SDS-PAGE, the membranes blotted with proteins were blocked with 4% BlockAce solution (Dainippon Pharmaceutical, Osaka, Japan) for 1 h and then incubated with antibodies against the following proteins: BIG3 (1:1,000); PHB2 (1:1,000, Abcam, Cambridge, UK); ERα (SP-1, 1:500, Thermo Fisher Scientific, Fremont, CA, USA); Akt, phospho-Akt (S473) (587F11, 1:1,000), p44/42 MAPK, phospho-p44/42 MAPK (T202/Y204) (1:1,000), VCP (valosin-containing protein) (1:500), and α/β-tubulin (1:1,000) (Cell Signaling Technology, Danvers, MA, USA); β-actin (AC-15; 1:5,000) and LMNB1 (1:100; Sigma) using standard procedures. All of the experiments were performed in triplicate at a minimum.