FIGURE 5.

Effect of REDD1 overexpression on 4E-BP1 binding to eIF4E and VEGF expression. TR-MUL cell cultures were maintained in DMEM containing 5 mm glucose and supplemented with 10% heat-inactivated FBS. Cells were transiently transfected with either an empty vector (EV) control plasmid or a plasmid encoding HA-tagged REDD1 (REDD1). A, binding of 4E-BP1 with eIF4E was assessed by co-immunoprecipitation. B, abundance of VEGF, HA-tagged REDD1, eIF4E, 4E-BP1, and tubulin was assessed in cell lysates by Western blot. SDS-PAGE was performed using 4–15% Tris-HCl polyacrylamide gels. Representative blots are shown. C, relative VEGF mRNA abundance was assessed by RT-qPCR. D, REDD1 expression was induced in TR-MUL cells after which cap-dependent and cap-independent translation was assessed using the bicistronic luciferase reporter described in 2F. Results represent the ratio of the relative LucF to LucR activity in TR-MUL cell lysate. REDD1 expression was induced by 6 h of doxycycline administration. Values are means + S.E. (n = 6). Statistical significance is denoted *, p < 0.05.