Ft synthesis exhibits a biphasic response during hypoxia. (A-B) HEK293 cells were grown for the indicated times under hypoxic conditions or under normoxia with Df (16 hours) or FAC. (A) Synthetic rate of Ft during hypoxia. For the last one hour of hypoxic or normoxic treatment, cells were labeled with 35S-Met/Cys in serum-free DMEM. Cell extracts were immunoprecipitated with rabbit antihuman Ft antibodies and proteins subjected to SDS-PAGE analysis and autoradiography. FAC, 50 μg/mL for 3 hours. Con indicates control immunoprecipitation lacking antibody. Experiment was performed 4 times with a representative blot shown. (B) Immunoblot analysis of cytosolic cell extracts with antihuman Ft antibodies. Df, 100 μM; FAC, 50 μg/mL for 5 hours. Experiment was performed 4 times with a representative blot shown. (C) Northern analysis of total cellular RNA. RNAs were transferred to a nylon membrane and hybridized with a 32P-labeled FtH, FtL, or 18S rRNA probe (control). Df, 200 μM; FAC, 150 μg/mL for 5 hours. Experiments were performed twice with representative blots shown.
