Fig. 1.

Analysis of the eIF4F translational complex in tamoxifen-sensitive and tamoxifen-resistant (TamR) MCF-7L breast carcinoma cells. a Cells were treated for 24 h with 100 nM tamoxifen or vehicle and then lysed using three freeze-thaw cycles. Equal amounts of protein were separated on an SDS-PAGE gel and immunoblotted with specific antibodies for the translational factors eIF4GI and eIF4E and the translational inhibitors 4E-BP2 and 4E-BP1. b MCF-7L (left) and T47D (right) cell lysates treated with either 100 nM tamoxifen or vehicle were incubated with 7-Me GTP Sepharose resin to capture the cap-binding protein eIF4E and its partners. The cap-bound material was eluted, subjected to SDS-PAGE, and immunoblotted. c Changes in integrity of the eIF4F complex in response to tamoxifen were quantified as differences between vehicle and tamoxifen treatments in eIF4GI/4E-BP1,2 relative density (R.D.) of the blots shown in b. d Plates of actively proliferating cells were treated for 24 h with 100 nM tamoxifen and then with cycloheximide (100 μg/mL) for 5 min followed by trypsinization and lysis. Cytoplasmic extract purified from the lysate was layered onto a sucrose gradient and then fractionated into ten, 1 mL fractions. Fractionated RNA was normalized to total RNA. e Cells transfected with a dual luciferase reporter construct (pcDNA3-rLuc-polIRES-fLuc) overnight. Transfection media were replaced with media containing 5% FBS and grown for another 24 h. Cells were then lysed, and levels of luciferase were read using a Dual-Luciferase Reporter Assay System (Promega)