Suppression of eIF4AI inhibits protein synthesis. (A) 35S-methionine/cysteine incorporation into TCA-insoluble protein. Following siRNA transfections, HeLa or HEK293 cells were allowed to recover for 48 h after which time they were labeled with 100 μCi/mL 35S-methionine/cysteine for 30 min. Cells were harvested, and the amount of radiolabeled protein was quantitated by TCA precipitation. The values presented are relative to cells transfected with siNT. Each value is the average of 10 independent experiments. Error bars, the SEM. Values are standardized against total protein content. (B) Reduction of global protein synthesis upon suppression of eIF4AI in HeLa cells. Metabolic labeling using 35S-methionine/cysteine and siRNA-treated HeLa cells was performed as in A. Equal protein amounts (15 μg) were resolved on a 10% polyacrylamide-SDS gel and electrophoresed, and the gel was treated with En3Hance, dried, and exposed to X-ray film (Kodak). (C) Polyribosome analysis of HeLa cells in which eIF4AI or eIF4AII was suppressed. All polysome profiles are from the same experiment performed on the same day. α-eIF4AI, sc-14211. (D) Western blot analysis of phospho-eIF2α status in HeLa cells. HeLa cells were transfected with the indicated siRNA for 48 h or treated with 10 μM arsenite for 15 min and harvested, and cell extracts were prepared. Protein samples (20 μg) were fractionated on an SDS-polyacrylamide gel, transferred to PVDF membrane, and probed with the indicated antibodies. α-eIF4AI, ab31217.
