Figure 3.

BCCIPβ is required for the ribosomal localization of S7 and cap-dependent/independent translations. (A) Cell extracts from control and BCCIPβ kd U2OS cells were fractionated through sucrose (20%) centrifugation. The proteins (S7, BCCIPα/β, and GAPDH) in nonribosome and ribosome fractions were detected by western blotting, and 18S rRNA was detected by electrophoresis. (B) HEK293T cells were transfected with wild-type or mutant Myc-S7 for 24 h. The proteins in nonribosome and ribosome fractions were separated and subjected to western blotting. (C) The newly synthesized proteins in control and BCCIPβ kd U2OS cells were labeled with AHA, click tagged with TAMRA, and analyzed by flow cytometry (left panel). The relative fluorescent intensity was determined (right panel). (D) Schematic representation of bicistronic reporter constructs with the indicated elements. (E) Control and BCCIPβ kd U2OS cells were transfected with the indicated bicistronic reporter plasmids, and Renilla and firefly luciferase activities in cell lysates were measured. (F) MCF7 cells were transfected with the bicistronic reporter construct (SV40/Ren/CrPV/FF) and BCCIPβ wild-type or mutant expression plasmids for 24 h, and Renilla and firefly luciferase activities in cell lysates were measured. The results are representative of at least three different experiments. *P < 0.05; **P < 0.01.