Inducible Knockdown of SHC1 in MCF7 Cells

MCF7 cells were cultured in Dulbecco’s modified Eagle’s medium (Wako Pure Chemical, Osaka, Japan) supplemented with 10% fetal bovine serum in a 5% CO₂ incubator at 37°C. These cultures were transfected with different expression vectors using previously described methods [32 (link)]. For the inducible knockdown of SHC, we used ON-TARGETplus Human SHC1 siRNA (Dharmacon, Lafayette, CO). After incubation at 37°C for 20 h, the cells were serum-starved in minimal essential medium (MEM; Nissui, Tokyo, Japan) containing 1.5 mg/mL NaHCO₃, 0.3 mg/mL L-glutamine, 15 mM HEPES (pH 7.4, Nacalai Tesque, Kyoto, Japan) and 0.1% fatty acid free BSA (Wako Pure Chemical). Cells were cultures in a 5% CO₂ incubator at 37°C for 24 h. To detect Halo-p52SHC and Halo-SHC3F expression, the cells were labeled with 100 nM HaloTag^® tetramethylrhodamine (TMR; Promega) in culture medium at 37°C for 15 min and washed repeatedly with HBSS (Sigma-Aldrich, St. Louis, MO). The medium was then replaced with MEM containing 5 mM HEPES (pH 7.4) and 0.1% BSA.