Figure 4
Expression of IGF-1R is critical for PDZK1 expression upon E2 stimulation in MCF-7 cells. (A) MCF-7 cells were treated with 1 nmol/L E2 for 6 h, after which cell lysates were prepared and incubated at 4°C overnight with a membrane containing fixed antibodies against the indicated growth factor. Bound growth factors were detected by enhanced chemiluminescence according to the manufacturer’s instructions. The relative signal intensity was quantified by using the VersaDoc imaging system. (B) MCF-7 cells were treated with 1 nmol/L E2 for different time intervals or left untreated. Total RNA was subjected to RT-PCR analysis with primers specific to human IGF-1R or β-actin. (C) MCF-7 cells were transiently transfected with siRNA targeting ER-α or control siRNA (Supplementary Figure S4). Cells were treated with 1 nmol/L E2 or left untreated for 48 h (left panel), after which protein extracts were prepared and subjected to immunoblot analysis with antibodies against IGF-1R or GAPDH. (D) MCF-7 cells were treated with 1 nmol/L E2 in the presence or absence of different doses of the IGF-1R antagonist AG1024 for 48 h or with human IGF-1 (10 ng/mL) for the indicated time intervals (right panel), after which protein extracts were prepared and subjected to immunoblot analysis with antibodies against PDZK1 or GAPDH.